Preparation of deallergenized proteins and permuteins

ABSTRACT

Modified proteins are disclosed that maintain enzymatic and insecticidal activity while displaying reduced or eliminated allergenicity. Epitopes which bind to anti-patatin antibodies were identified, and removed via site directed mutagenesis. Tyrosines were observed to generally contribute to the allergenic properties of palatin proteins. Removal of glycosylation sites was observed to reduce or eliminate antibody binding. Permuteins are also disclosed which have a rearranged amino acid sequence while retaining enzymatic activity. Deallergenized proteins and permuteins can be used as insecticidal materials, as nutritional supplements, and as immunotherapeutic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 09/755,630 filed Jan. 5, 2001 now U.S. Pat. No. 6,639,054,which claims the benefit of U.S. Provisional Application Ser. No.60/174,669 filed on Jan. 6, 2000.

FIELD OF THE INVENTION

The invention relates generally to non-naturally occurring novelproteins which have insecticidal properties, and more specifically tothe design, preparation, and use of proteins that have beendeallergenized while maintaining their insecticidal properties.Deallergenized patatin proteins include variants that have hadallergenic sequences modified, and permuteins that have had their aminoacid sequences rearranged at one or more breakpoints.

BACKGROUND OF THE INVENTION

Insecticidal Proteins

The use of natural products, including proteins, is a well known methodof controlling many insect, fungal, viral, bacterial, and nematodepathogens. For example, endotoxins of Bacillus thuringiensis (B.t. ) areused to control both lepidopteran and coleopteran insect pests. Genesproducing these endotoxins have been introduced into and expressed byvarious plants, including cotton, tobacco, and tomato. There are,however, several economically important insect pests such as boll weevil(BWV), Anthonomus grandis, and corn rootworm (CRW), Diabrotica spp. thatare not as susceptible to B.t. endotoxins as are insects such aslepidopterans. In addition, having other, different gene products forcontrol of insects which are susceptible to B.t. endotoxins isimportant, if not vital, for resistance management.

It has been recently discovered that the major storage protein of potatotubers, patatins (Gaillaird, T., Biochem. J. 121: 379-390, 1971;Racusen, D., Can. J. Bot., 62: 1640-1644, 1984; Andrews, D. L., et al.,Biochem. J., 252: 199-206, 1988), will control various insects,including western rootworm (WCRW, Diabrotica virigifera), southern cornrootworm (SCRW, Diabrotica undecimpunctata), and boll weevil (BWV,Anthonomus grandis) (U.S. Pat. No. 5,743,477). Patatins are lethal tosome larvae and will stunt the growth of survivors so that maturation isprevented or severely delayed, resulting in no reproduction. Theseproteins, have nonspecific lipid acyl hydrolase activity and studieshave shown that the enzyme activity is essential for its insecticidalactivity (Strickland, J. A., et al., Plant Physiol., 109: 667-674, 1995;U.S. Pat. No. 5,743,477). Patatins can be applied directly to the plantsor introduced in other ways well known in the art, such as through theapplication of plant-colonizing microorganisms, which have beentransformed to produce the enzymes, or by the plants themselves aftersimilar transformation.

In potato, the patatins are found predominantly in tubers, but also atmuch lower levels in other plant organs (Hofgen, R. and Willmitzer, L.,Plant Science, 66: 221-230, 1990). Genes that encode patatins have beenpreviously isolated by Mignery, G. A., et al. (Nucleic Acids Research,12: 7987-8000, 1984; Mignery, G. A., et al., Gene, 62: 27-44, 1988;Stiekema, et al., Plant Mol. Biol., 11: 255-269, 1988) and others.Patatins are found in other plants, particularly solanaceous species(Ganal, et al., Mol. Gen. Genetics, 225: 501-509, 1991; Vancanneyt, etal., Plant Cell, 1: 533-540, 1989) and recently Zea mays (WO 96/37615).Rosahl, et al. (EMBO J., 6: 1155-1159, 1987) transferred it to tobaccoplants, and observed expression of patatin, demonstrating that thepatatin genes can be heterologously expressed by plants.

Patatin is an attractive for use in planta as an insect control agent,but unfortunately a small segment of the population displays allergicreactions to patatin proteins, and in particular to potato patatin, asdescribed below.

Food Allergens

There are a variety of proteins that cause allergic reactions. Proteinsthat have been identified as causing an allergic reaction inhypersensitive patients occur in many plant and animal derived foods,pollens, fungal spores, insect venoms, insect feces, and animal danderand urine (King, H. C., Ear Nose Throat J., 73(4): 237-241, 1994;Astwood, J. D., et al., Clin. Exp. Allergy, 25: 66-72, 1995; Astwood, J.D. and Fuchs R. L., Monographs in allergy Vol. 32: Highlights in foodallergy, pp. 105-120, 1996; Metcalfe, D. D., et al., Critical Reviews inFood Science and Nutrition, 36S: 165-186, 1996). The offending proteinsof many major sources of allergens have been characterized by clinicaland molecular methods. The functions of allergenic proteins in vivo arediverse, ranging from enzymes to regulators of the cell cytoskeleton.

To understand the molecular basis of allergic disease, the important IgEbinding epitopes of many allergen proteins have been mapped (Elsayed, S.and Apold, J., Allergy 38(7): 449-459, 1983; Elsayed, S. et al., ScandJ. Clin. Lab. Invest. Suppl. 204: 17-31 1991; Zhang, L., et al., Mol.Immunol. 29(11): 1383-1389, 1992). The optimal peptide length for IgEbinding has been reported to be between 8 and 12 amino acids.Conservation of epitope sequences is observed in homologous allergens ofdisparate species (Astwood, J. D., et al., Clin. Exp. Allergy, 25:66-72, 1995). Indeed, conservative substitutions introduced bysite-directed mutagenesis reduce IgE binding of known epitopes whenpresented as peptides.

Food allergy occurs in 2-6% of the population. Eight foods or foodgroups (milk, eggs, fish, crustacea, wheat, peanuts, soybeans, and treenuts) account for 90% of allergies to foods. Nevertheless, over 160different foods have been reported to cause adverse reactions, includingpotato (Hefle, S., et al., Crit. Rev. in Food Sci. Nutr., 36S: 69-90,1996).

Mode of Action of Allergens

Regardless of the identity of the allergen, it is theorized that theunderlying mechanism of allergen response is the same. Immediatehypersensitivity (or anaphylactic response) is a form of allergicreaction which develops very quickly, i.e., within seconds or minutes ofexposure of the patient to the causative allergen, and is mediated by Blymphocyte IgE antibody produciton. Allergic patients exhibit elevatedlevels of IgE, mediating hypersensitivity by priming mast cells whichare abundant in the skin, lymphoid organs, in the membranes of the eye,nose and mouth, and in the respiratory tree and intestines. The IgE inallergy-suffering patients becomes bound to the IgE receptors of mastcells. When this bound IgE is subsequently contacted by the appropriateallergen, the mast cell is caused to degranulate and release varioussubstances such as histamine into the surrounding tissue (Church et al.In: Kay, A. B. ed., Allergy and Allergic Diseases, Oxford, BlackwellScience, pp. 149-197, 1997).

It is the release of these substances which is responsible for theclinical symptoms typical of immediate hypersensitivity, namelycontraction of smooth muscle in the airways or in the intestine, thedilation of small blood vessels, and the increase in their permeabilityto water and plasma proteins, the secretion of thick sticky mucus, and(in the skin) the stimulation of nerve endings that result in itching orpain. Immediate hypersensitivity is, at best, a nuisance to the suffer;at worst it can present very serious problems and can in rare cases evenresult in death.

Allergic Reactions to Potato

Food allergy to potato is considered rare in the general population(Castells, M. C., et al., Allergy Clin. Immunol., 8: 1110-1114, 1986;Hannuksela, M., et al., Contact Dermatitis, 3: 79-84, 1977; Golbert, T.M., et al., Journal of Allergy, 44: 96-107, 1969). Approximately 200individuals have participated in published clinical accounts of potatoallergy (Hefle, S. et al., Critical Reviews in Food Science andNutrition, 36S: 69-90, 1996). A number of IgE binding proteins have beenidentified in potato tuber extracts (see Table 1), however the aminoacid sequence and function of these proteins has not been determined(Wahl, R., et al., Intl. Arch. Allergy Appl. Immunol., 92: 168-174,1990).

TABLE 1 Studies of potato tuber IgE-binding proteins (allergens) StudyProtein Characteristics (Castells, M. C. et al. J. Allergy Clin. Unknown14 to 40 kDa Immunol. 78, 1110-1114, 1986) (Wahl, R. et al. Int. Arch.Allergy Appl. Unknown 42/43 kDa Immunol. 92: 168-174, 1990) Unknown 65kDa Unknown 26 kDa Unknown 20 kDa Unknown 14 kDa Unknown <14 kDa (˜5kDa) (Ebner, C. et al. in: Wuthrich, B. & Unknown 42/43 kDa Ortolani, C.(eds.), Highlights in food allergy. Monographs in Allergy, Volume 32Basil, Karger, pp. 73-77, 1996) Unknown 23 kDa Unknown ˜16 kDa Unknown<14 kDa (˜5 kDa)

Improved Safety from the use of Hypoallergenic Proteins

Patatin has been identified as an allergenic protein (Seppala, U. etal., J. Allergy Clin. Immunol. 103:165-171, 1999). Accordingly, potatoallergic subjects may not be able to safely consume products containingunmodified patatin protein, such as crops to which foliar applicationsof patatins have been applied, or crops which have been engineered toexpress patatin. In addition, proliferation of food allergens in thefood supply is considered hazardous (Metcalfe, D. D., et al., CriticalReviews and Food Science and Nutrition, 36S: 165-186, 1996). There areadditional concerns regarding the use of potentially allergenic foodproteins where workers might be exposed to airborne particulates,initiating a new allergic response (Moneret-Vautrin, D. A., et al., Rev.Med. Interne., 17(7): 551-557, 1996).

Permuteins

Novel proteins generated by the method of sequence transpositionresembles that of naturally occurring pairs of proteins that are relatedby linear reorganization of their amino acid sequences (Cunningham, etal. Proc. Natl. Sci., U.S.A., 76: 3218-3222, 1979; Teather, et al., J.Bacteriol., 172: 3837-3841, 1990; Schimming, et al., Eur. J. Biochem.,204: 13-19, 1992; Yamiuchi, et al., FEBS Lett., 260: 127-130, 1991;MacGregor, et al., FEBS. Lett., 378: 263-266, 1996). The first in vitroapplication of sequence rearrangement to proteins was described byGoldenberg and Creighton (Goldenberg and Creighton, J. Mol. Biol., 165:407-413, 1983). A new N-terminus is selected at an internal site(breakpoint) of the original sequence, the new sequence having the sameorder of amino acids as the original from the breakpoint until itreaches an amino acid that is at or near the original C-terminus. Atthis point the new sequence is joined, either directly or through anadditional portion or sequence (linker), to an amino acid that is at ornear the original N-terminus, and the new sequence continues with thesame sequence as the original until it reaches a point that is at ornear the amino acid that was N-terminal to the breakpoint site of theoriginal sequence, this residue forming the new C-terminus of the chain.This approach has been applied to proteins which range in size from 58to 462 amino acids and represent a broad range of structural classes(Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983; Li andCoffino, Mol. Cell. Biol., 13: 2377-2383, 1993; Zhang, et al., NatureStruct. Biol., 1: 434-438, 1995; Buchwalder, et al., Biochemistry, 31:1621-1630, 1994; Protasova, et al., Prot. Eng., 7: 1373-1377, 1995;Mullins, et al., J. Am. Chem. Soc., 116: 5529-5533, 1994; Garrett, etal., Protein Science, 5: 204-211, 1996; Hahn, et al., Proc. Natl. Acad.Sci. USA., 91: 10417-10421, 1994; Yang and Schachman, Proc. Natl. Acad.Sci. U.S.A., 90: 11980-11984, 1993; Luger, et al., Science, 243:206-210, 1989; Luger, et al., Prot. Eng., 3: 249-258, 1990; Lin, et al.,Protein Science, 4: 159-166, 1995; Vignais, et al., Protein Science, 4:994-1000, 1995; Ritco-Vonsovici, et al., Biochemistry, 34: 16543-16551,1995; Horlick, et al., Protein Eng., 5: 427-431, 1992; Kreitman, et al.,Cytokine, 7: 311-318, 1995; Viguera, et al., Mol. Biol., 247: 670-681,1995; Koebnik and Kramer, J. Mol. Biol., 250: 617-626, 1995; Kreitman,et al., Proc. Natl. Acad. Sci., 91: 6889-6893, 1994).

There exists a need for the development of plant expressibleinsecticidal proteins which possess minimal or no allergenic properties.

SUMMARY OF THE INVENTION

Novel protein sequences, and nucleic acid sequences encoding them aredisclosed. The proteins maintain desirable enzymatic and insecticidalproperties while displaying reduced or eliminated allergenicity.

Allergenic epitopes are identified by scanning overlapping peptidesequences with an immunoreactivity assay. Alanine scanning and ‘rationalsubstitution’ is performed on identified peptide sequences to determinespecific amino acids which contribute to antibody binding, andpresumably, to the allergenic properties of the whole protein.Individual mutations are introduced into the whole protein sequence bymethods such as site directed mutagenesis of the encoding nucleic acidsequence to delete or modify the allergenic sequences.

Glycosylation target residues are identified within amino acid sequencesof proteins which have demonstrated allergy eliciting properties.Glycosylation target amino acid residues are rationally substutited withother amino acid residues to eliminate glycosylation and to provide avariant deglycosylated protein. The variant protein may then exhibitreduced allergen eliciting properties and may also exhibit reducedbinding to IgE within serum of patients observed to be allergic to saidglycosylated protein.

Permuteins of the deallergenized protein sequences can be constructed tofurther reduce or eliminate allergic reactions. The encoding nucleicacid sequence is modified to produce a non-naturally occurring proteinhaving a linear amino acid sequence different from the naturallyoccurring protein sequence, while maintaining enzymatic and insecticidalproperties. The permutein is preferably produced in plant cells, andmore preferably produced at a concentration which is toxic to insectsingesting the plant cells.

Methods for reducing, eliminating, or decreasing allergen elicitingproperties of a protein are specifically contemplated herein. Suchmethods comprise steps including identifying one or more patientsexhibiting an allergic sensitivity to an allergen eliciting protein andobtaining a sample of serum from the patient; exposing the patient serumto a first set of synthetic overlapping peptides which represent theallergen eliciting protein in order to identify such peptides whichexhibit epitopes which bind to IgE present within the allergic patients'serum and wherein the IgE present in the serum has a specific affinityfor the said allergen eliciting protein; producing a second set ofpeptides which are variant peptides based on the first set of peptideswhich were identified to bind specifically to IgE present in patientserum, wherein the second set variant peptides exhibit alanine scanningor rational scanning amino acid substitutions which exhibit reduced,decreased, or eliminated IgE binding when compared to the first setnon-variant peptides, and wherein such substitutions which reduce,eliminate or decrease IgE binding are identified as result effectivesubstitutions; and modifying the amino acid sequence of the allergeneliciting protein to contain one or more of said result effectivesubstitutions, wherein the modified protein is a variant of the allergeneliciting protein which lacks allergen eliciting protein or exhibitsreduced allergen eliciting properties, and wherein the variant of theallergen eliciting protein comprising one or more result effectivesubstitutions exhibits reduced, decreased, or totally eliminated bindingof IgE present within said patients' serum.

The novel proteins can be used in controlling insects, as nutritionalsupplements, in immunotherapy protocols, and in other potentialapplications. Transgenic plant cells and plants containing the encodingnucleic acid sequence can be particularly beneficial in the control ofinsects, and as a nutritional/immunotherapy material.

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention can be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 illustrates the alignment of potato patatin PatA (acyl lipidhydrolase) with patatin (acyl lipid hydrolase) homologs and relatedamino acid sequences, the homologs and related sequences being from bothdicot and monocot plant species.

FIG. 2 illustrates IgE binding to overlapping peptide sequences.

FIG. 3 illustrates construction of nucleic acid sequences encodingpatatin permutein proteins, and in this figure for illustrative purposesa breakpoint at position 247 is shown.

DESCRIPTION OF THE SEQUENCE LISTINGS

The following description of the sequence listing forms part of thepresent specification and is included to further demonstrate certainaspects of the present invention. The invention can be better understoodby reference to one or more of these sequences in combination with thedetailed description of specific embodiments presented herein.

SEQ ID NO: 1 DNA sequence encoding a patatin (acyl lipid hydrolase)protein SEQ ID NO: 2 potato patatin protein sequence SEQ ID NO: 3thermal amplification primer SEQ ID NO: 4 thermal amplification primerSEQ ID NO: 5 thermal amplification product SEQ ID NO: 6 Pre-cleavagepatatin protein produced in Pichia pastoris SEQ ID NO: 7 Post-cleavagepatatin protein produced in Pichia pastoris SEQ ID NO: 8 Y106F mutagenicprimer SEQ ID NO: 9 Y129F mutagenic primer SEQ ID NO: 10 Y185F mutagenicprimer SEQ ID NO: 11 Y193F mutagenic primer SEQ ID NO: 12 Y185F andY193F mutagenic primer SEQ ID NO: 13 Y270F mutagenic primer SEQ ID NO:14 Y316F mutagenic primer SEQ ID NO: 15 Y362F mutagenic primer SEQ IDNO: 16-104 Peptide scan sequences of a patatin protein SEQ ID NO:105-241 Alanine and rational scan sequences of selected patatin peptidesSEQ ID NO: 242 thermal amplification primer 27 SEQ ID NO: 243 thermalamplification primer 48 SEQ ID NO: 244 thermal amplification primer 47SEQ ID NO: 245 thermal amplification primer 36 SEQ ID NO: 246 pMON37402sequence encoding permutein protein SEQ ID NO: 247 Permutein proteinencoded from pMON37402 sequence SEQ ID NO: 248 thermal amplificationprimer 58 SEQ ID NO: 249 thermal amplification primer 59 SEQ ID NO: 250pMON37405 sequence encoding permutein protein SEQ ID NO: 251 Permuteinprotein encoded by pMON37405 sequence SEQ ID NO: 252 thermalamplification primer 60 SEQ ID NO: 253 thermal amplification primer 61SEQ ID NO: 254 pMON37406 sequence encoding permutein protein SEQ ID NO:255 Permutein protein encoded by pMON37406 sequence SEQ ID NO: 256thermal amplification primer 62 SEQ ID NO: 257 thermal amplificationprimer 63 SEQ ID NO: 258 pMON37407 sequence encoding permutein proteinSEQ ID NO: 259 Permutein protein encoded by pMON37407 sequence SEQ IDNO: 260 thermal amplification primer 60 SEQ ID NO: 261 thermalamplification primer 65 SEQ ID NO: 262 pMON37408 sequence encodingpermutein protein SEQ ID NO: 263 Permutein protein encoded by pMON37408sequence SEQ ID NO: 264 pMON40701 sequence encoding permutein proteinSEQ ID NO: 265 Permutein protein encoded by pMON40701 sequence SEQ IDNO: 266 thermal amplification primer Syn1 SEQ ID NO: 267 thermalamplification primer Syn2 SEQ ID NO: 268 thermal amplification primerSyn3 SEQ ID NO: 269 thermal amplification primer Syn4 SEQ ID NO: 270pMON40703 sequence encoding permutein protein SEQ ID NO: 271 Permuteinprotein encoded by pMON40703 sequence SEQ ID NO: 272 thermalamplification primer Syn10 SEQ ID NO: 273 thermal amplification primerSyn11 SEQ ID NO: 274 pMON40705 sequence encoding permutein protein SEQID NO: 275 Permutein protein encoded by pMON40705 sequence SEQ ID NO:276-277 Permutein linker sequences SEQ ID NO: 278 Patatin isozyme PatA+(including signal peptide) SEQ ID NO: 279 Patatin isozyme PatB+(including signal peptide) SEQ ID NO: 280 Patatin isozyme PatFm (matureprotein lacking signal peptide) SEQ ID NO: 281 Patatin isozyme PatIm(mature protein lacking signal peptide) SEQ ID NO: 282 Patatin isozymePatL+ (including signal peptide) SEQ ID NO: 283 Rational substitutionpeptide SEQ ID NO: 284 Corn homolog peptide SEQ ID NO: 285 patatinhomolog Pat17 DNA coding sequence and amino acid translation SEQ ID NO:286 patatin homolog Pat17 amino acid sequence SEQ ID NO: 287 dicotpatatin homolog amino acid sequence pentin1_phb SEQ ID NO: 288 dicotpatatin homolog amino acid sequence 5c9_phb SEQ ID NO: 289 maize patatinhomolog amino acid sequence corn1_pep SEQ ID NO: 290 maize patatinhomolog amino acid sequence corn2_pep SEQ ID NO: 291 maize patatinhomolog amino acid sequence corn3_pep SEQ ID NO: 292 maize patatinhomolog amino acid sequence corn4_pep SEQ ID NO: 293 maize patatinhomolog amino acid sequence corn5_pep

Definitions

The following definitions are provided in order to aid those skilled inthe art in understanding the detailed description of the presentinvention. Some words and phrases may also be defined in other sectionsof the specification. No limitation should be placed on the definitionspresented for the terms below, where other meanings are evidencedelseqhere in the specification in addition to those specified below.

“Allergen” refers to a biological or chemical substance that induces anallergic reaction or response. An allergic response can be animmunoglobulin E-mediated response.

Amino acid codes: A (Ala)=alanine; C (Cys)=cysteine; D (Asp)=asparticacid; E (Glu)=glutamic acid; F (Phe)=phenylalanine; G (Gly)=glycine; H(His)=histidine; I (Ile)=isoleucine; K (Lys)=lysine; L (Leu)=leucine; M(Met)=methionine; N (Asn)=asparagine; P (Pro)=proline; Q(Gln)=glutamine; R (Arg)=arginine; S (Ser)=serine; T (Thr)=threonine;V=(Val) valine; W (Trp)=tryptophan; Y (Tyr)=tyrosine.

“Amplification: refers to increasing the number of copies of a desiredmolecule.

“Coding sequence”, “open reading frame”, and “structural sequence” referto the region of continuous sequential nucleic acid base pair tripletsencoding a protein, polypeptide, or peptide sequence.

“Codon” refers to a sequence of three nucleotides that specify aparticular amino acid.

“Complementarity” refers to the specific binding of adenine to thymine(or uracil in RNA) and cytosine to guanine on opposite strands of DNA orRNA.

“Deallergenize” (render hypoallergenic) refers to the method ofengineering or modifying a protein or the encoding DNA such that theprotein has a reduced or eliminated ability to induce an allergicresponse with respect to the ability of the unmodified protein. Adeallergenized protein can be referred to as being hypoallergenic. Thedegree of deallergenization of a protein can be measured in vitro by thereduced binding of IgE antibodies.

“DNA segment heterologous to the promoter region” means that the codingDNA segment does not exist in nature in the same gene with the promoterto which it is now attached.

“DNA segment” refers to a DNA molecule that has been isolated free oftotal genomic DNA of a particular species.

“Electroporation” refers to a method of introducing foreign DNA intocells that uses a brief, high voltage DC (direct current) charge topermeabilize the host cells, causing them to take up extra-chromosomalDNA.

“Encoding DNA” refers to chromosomal DNA, plasmid DNA, cDNA, orsynthetic DNA which encodes any of the enzymes discussed herein.

“Endogenous” refers to materials originating from within an organism orcell.

“Endonuclease” refers to an enzyme that hydrolyzes double stranded DNAat internal locations.

“Epitope” refers to a region on an allergen that interacts with thecells of the immune system. Epitopes are often further defined by thetype of antibody or cell with which they interact, e.g. if the regionreacts with B-cells or antibodies (IgE), it is called a B-cell epitope.

“Exogenous” refers to materials originating from outside of an organismor cell. This typically applies to nucleic acid molecules used inproducing transformed or transgenic host cells and plants.

“Expressibly coupled” and “expressibly linked” refer to a promoter orpromoter region and a coding or structural sequence in such anorientation and distance that transcription of the coding or structuralsequence can be directed by the promoter or promoter region.

“Expression” refers to the transcription of a gene to produce thecorresponding mRNA and translation of this mRNA to produce thecorresponding gene product, i.e., a peptide, polypeptide, or protein.

“Heterologous DNA” refers to DNA from a source different than that ofthe recipient cell.

“Homologous DNA” refers to DNA from the same source as that of therecipient cell.

“Identity” refers to the degree of similarity between two nucleic acidor protein sequences. An alignment of the two sequences is performed bya suitable computer program. A widely used and accepted computer programfor performing sequence alignments is CLUSTALW v1.6 (Thompson, et al.Nucl. Acids Res., 22: 4673-4680, 1994). The number of matching bases oramino acids is divided by the total number of bases or amino acids, andmultiplied by 100 to obtain a percent identity. For example, if two 580base pair sequences had 145 matched bases, they would be 25 percentidentical. If the two compared sequences are of different lengths, thenumber of matches is divided by the shorter of the two lengths. Forexample, if there were 100 matched amino acids between 200 and a 400amino acid proteins, they are 50 percent identical with respect to theshorter sequence. If the shorter sequence is less than 150 bases or 50amino acids in length, the number of matches are divided by 150 (fornucleic acid bases) or 50 (for amino acids), and multiplied by 100 toobtain a percent identity.

“IgE” (Immunoglobulin E) refers to a specific class of immunoglobulinsecreted by B cells. IgE binds to specific receptors on Mast cells.Interaction of an allergen with mast cell-bound IgE may trigger allergicsymptoms.

“Immunotherapy” refers to any type of treatment that targets the immunesystem. Allergy immunotherapy is a treatment in which a progressivelyincreasing dose of an allergen is given in order to induce an immuneresponse characterized by tolerance to the antigen/allergen, also knownas desensitization.

“In vitro” refers to “in the laboratory” and/or “outside of a livingorganism”.

“In vivo” refers to “in a living organism”.

“Insecticidal polypeptide” refers to a polypeptide having insecticidalproperties that adversely affects the growth and development of insectpests.

“Monocot” refers to plants having a single cotyledon (the first leaf ofthe embryo of seed plants); examples include cereals such as maize,rice, wheat, oats, and barley.

“Multiple cloning site” refers to an artificially constructed collectionof restriction enzyme sites in a vector that facilitates insertion offoreign DNA into the vector.

“Mutation” refers to any change or alteration in a nucleic acidsequence. Several types exist, including point, frame shift, splicing,and insertion/deletions.

“Native” refers to “naturally occurring in the same organism”. Forexample, a native promoter is the promoter naturally found operativelylinked to a given coding sequence in an organism. A native protein isone naturally found in nature and untouched or not otherwise manipulatedby the hand of man.

“Nucleic acid segment” is a nucleic acid molecule that has been isolatedfree of total genomic DNA of a particular species, or that has beensynthesized. Included with the term “nucleic acid segment” are DNAsegments, recombinant vectors, plasmids, cosmids, phagemids, phage,viruses, etcetera.

“Nucleic acid” refers to deoxyribonucleic acid (DNA) and ribonucleicacid (RNA).

Nucleic acid codes: A=adenosine; C=cytosine; G=guanosine; T=thymidine;N=equimolar A, C, G, and T; I=deoxyinosine; K=equimolar G and T;R=equimolar A and G; S=equimolar C and G; W=equimolar A and T;Y=equimolar C and T.

“Open reading frame (ORF)” refers to a region of DNA or RNA encoding apeptide, polypeptide, or protein or capable of being translated toprotein, or a regioiu of DNA capable of being transcribed into an RNAproduct.

“Plasmid” refers to a circular, extrachromosomal, self-replicating pieceof DNA.

“Point mutation” refers to an alteration of a single nucleotide in anucleic acid sequence.

“Polymerase chain reaction (PCR)” refers to an enzymatic technique tocreate multiple copies of one sequence of nucleic acid. Copies of DNAsequence are prepared by shuttling a DNA polymerase between twooligonucleotides. The basis of this amplification method is multiplecycles of temperature changes to denature, then re-anneal amplimers,followed by extension to synthesize new DNA strands in the regionlocated between the flanking amplimers. Also known as thermalamplification.

“Probe” refers to a polynucleotide sequence which is complementary to atarget polynucleotide sequence in the analyte. An antibody can also beused as a probe to detect the presence of an antigen. In that sense, theantigen binding domain of the antibody has some detectable affinity forthe antigen and binds thereto. The binding of the antibody to theantigen can be measured by means known in the art, such as bychemiluminescence, phosphorescence, flourescence, colorimetric chemicaldeposition at the site of binding, or otherwise.

“Promoter” or “promoter region” refers to a DNA sequence, usually foundupstream (5′) to a coding sequence, that controls expression of thecoding sequence by controlling production of messenger RNA (mRNA) byproviding the recognition site for RNA polymerase and/or other factorsnecessary for start of transcription at the correct site. Ascontemplated herein, a promoter or promoter region includes variationsof promoters derived by means of ligation to various regulatorysequences, random or controlled mutagenesis, and addition or duplicationof enhancer sequences. The promoter region disclosed herein, andbiologically functional equivalents thereof, are responsible for drivingthe transcription of coding sequences under their control whenintroduced into a host as part of a suitable recombinant vector, asdemonstrated by its ability to produce mRNA.

“Recombinant DNA construct” or “recombinant vector” refers to any agentsuch as a plasmid, cosmid, virus, autonomously replicating sequence,phage, or linear or circular single-stranded or double-stranded DNA orRNA nucleotide sequence, derived from any source, capable of genomicintegration or autonomous replication, comprising a DNA molecule inwhich one or more DNA sequences have been linked in a functionallyoperative manner. Such recombinant DNA constructs or vectors are capableof introducing a 5′ regulatory sequence or promoter region and a DNAsequence for a selected gene product into a cell in such a manner thatthe DNA sequence is transcribed into a functional mRNA which istranslated and therefore expressed. Recombinant DNA constructs orrecombinant vectors can be constructed to be capable of expressingantisense RNAs, in order to inhibit translation of a specific RNA ofinterest.

“Recombinant proteins”, also referred to as “heterologous proteins”, areproteins which are normally not produced by the host cell.

“Regeneration” refers to the process of growing a plant from a plantcell (e.g., plant protoplast or explant).

“Regeneration” refers to the process of growing a plant from a plantcell (e.g., plant protoplast or explant).

“Regulatory sequence” refers to a nucleotide sequence located upstream(5′), within, and/or downstream (3′) to a DNA sequence encoding aselected gene product whose transcription and expression is controlledby the regulatory sequence in conjunction with the protein syntheticapparatus of the cell.

“Restriction enzyme” refers to an enzyme that recognizes a specificpalindromic sequence of nucleotides in double stranded DNA and cleavesboth strands; also called a restriction endonuclease. Cleavage typicallyoccurs within the restriction site.

“Result-effective substitution” (RES) refers to an amino acidsubstitution within an IgE-binding region (epitope) of a target proteinwhich reduces or eliminates the IgE binding by that epitope. Examplesherein are directed to patatin protein and homologues, however, as willbe readily recognized by those skilled in the art, the method is morebroadly applicable to proteins other than patatins, and in particular isapplicable to any protein exhibiting allergen eliciting properties.

“Selectable marker” refers to a nucleic acid sequence whose expressionconfers a phenotype facilitating identification of cells containing thenucleic acid sequence. Selectable markers include those which conferresistance to toxic chemicals (e.g. ampicillin resistance, kanamycinresistance), complement a nutritional deficiency (e.g. uracil,histidine, leucine), or impart a visually distinguishing characteristic(e.g. color changes or fluorescence).

“Transcription” refers to the process of producing an RNA copy from aDNA template.

“Transformation” refers to a process of introducing an exogenous nucleicacid sequence (e.g., a vector, recombinant nucleic acid molecule) into acell or protoplast in which that exogenous nucleic acid is incorporatedinto a chromosome or is capable of autonomous replication.

“Transformed cell” is a cell whose DNA has been altered by theintroduction of an exogenous nucleic acid molecule into that cell.

“Transgenic cell” refers to any cell derived from or regenerated from atransformed cell or derived from a transgenic cell. Exemplary transgeniccells include plant calli derived from a transformed plant cell andparticular cells such as leaf, root, stem, e.g., somatic cells, orreproductive (germ) cells obtained from a transgenic plant.

“Transgenic plant” refers to a plant or progeny thereof derived from atransformed plant cell or protoplast, wherein the plant DNA contains anintroduced exogenous nucleic acid sequence not originally present in anative, non-transgenic plant of the same species. Alternatively, theplant DNA can contain the introduced nucleic acid sequence in a highercopy number than in the native, non-transgenic plant of the samespecies.

“Translation” refers to the production of protein from messenger RNA.

“Vector” refers to a plasmid, cosmid, bacteriophage, or virus thatcarries foreign DNA into a host organism.

“Western blot” refers to protein or proteins that have been separated byelectrophoresis, transferred and immobilized onto a solid support, thenprobed with an antibody.

DETAILED DESCRIPTION OF THE INVENTION

Design of Deallergenized Patatin Proteins

Deallergenizing a protein can be accomplished by the identification ofallergenic sites, followed by modification of the sites to reduce oreliminate the binding of antibodies to the sites. The IgE-bindingregions of patatin were previously unreported. Mapping of the IgEepitopes was accomplished by synthesizing 10-mer peptides based on thepatatin 17 protein sequence (SEQ ID NO: 2) which overlap by six aminoacids. As potato proteins are denatured upon cooking potato products, itis expected that the 10-mer peptides sufficiently mimic the unfoldedfull length protein for antibody binding purposes. Peptides wereidentified based upon their ability to bind to IgE antibodies.Individual amino acids within the identified peptides were changed toreduce or eliminate binding to IgE present in sera from potato sensitivepatients. These changes are termed result-effective amino acidsubstitutions (RES). The RES can be subsequently introduced into thefull length protein by site directed mutagenesis of the encoding nucleicacid sequence or other means known in the art. Similar strategies havebeen employed elsewhere to determine the dominant IgE epitopes in amajor peanut allergen (Stanley, J. S., et al., Arch. Biochem. Biophys.,342(2): 244-253, 1997).

Certain amino acid residues important for allergenicity of patatin areidentified. Some of the designed patatin peptides wherein single aminoacid residues were replaced with alanine or phenylalanine, showedsignificantly reduced or no binding to sera from potato sensitivepatients.

A “deallergenized patatin” refers to a patatin protein differing in atleast one of the amino acid residues as defined by the result effectivesubstitutions resulting in the patatin protein having reduced reactivitytowards sera from potato sensitive patients. The deallergenized patatinpreferably maintains insecticidal properties, and preferably maintainsits characteristic enzymatic profile.

Summary of Method to Deallergenize a Patatin Protein

-   -   Mapping of IgE epitopes by immunoassay of synthetic overlapping        peptides using sera from potato sensitive patients;    -   Identification of result-effective substitutions by alanine        scanning and/or rational scanning;    -   Modification of the amino acid sequence of patatin by        site-directed mutagenesis of the encoding nucleic acid sequence;    -   Evaluation of enzyme activity (esterase) and/or insecticidal        activity of the modified protein(s); and    -   Evaluation of the new protein(s) for allergenicity by IgE        immunoassay.

Nucleic acid sequences encoding patatin have been cloned by severalinvestigators (e.g. Mignery, et al., Nucleic Acids Research, 12:7987-8000, 1984; Mignery, et al., Gene, 62: 27-44, 1988; WO 94/21805;Canadian Patent Application No. 2090552). These nucleic acid sequencescan then be manipulated using site directed mutagenesis to encode ahypoallergenic patatin. These nucleic acid sequences can than be used totransform bacterial, yeast or plant cells, resulting in the productionof hypoallergenic patatin protein.

Deallergenized Patatin Proteins

For simplicity, individual amino acids are referred to by their singleletter codes. Correlation between the single letter codes, three lettercodes, and full amino acid names is presented in the definitions sectionabove.

One embodiment of the invention is an isolated deallergenized patatinprotein. The protein is modified relative to the wild-type proteinsequence such that they exhibit reduced binding to anti-patatinantibodies such as those obtained from humans or animals allergic topotatoes. The reduced binding is measured relative to the binding of theunmodified patatin protein to the anti-patatin antibodies.

The deallergenized patatin protein can comprise SEQ ID NO:2 modified inone or more of the following regions, or SEQ ID NO:7 modified in one ormore of the following regions. The single or multiple amino acidmodifications reduce the binding of the modified protein relative to thebinding of the corresponding unmodified protein. The regions formodification include amino acid positions 104-113 of SEQ ID NO:2 (85-94of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7),184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2(245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ IDNO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possibleamino acid modifications include replacing an amino acid with A, E, F,P, or S. The modifications replace one or more amino acids in theidentified regions, without increasing or decreasing the total number ofamino acids in the protein.

Preferably, the deallergenized patatin protein comprises SEQ ID NO:2modified by one or more changes, or SEQ ID NO:7 modified by one or morechanges. SEQ ID NO:7 differs from wild type SEQ ID NO:2 in that thefirst 22 amino acids of SEQ ID NO:2 are replaced with EAE (Glu-Ala-Glu).For example, the changes to SEQ ID NO:2 or SEQ ID NO:7 can be: the Ycorresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ IDNO:7 is replaced with F or A; the I corresponding to position 113 of SEQID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 137 of SEQID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the Scorresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 185 of SEQ IDNO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the Acorresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ IDNO:7 is replaced with S; the T corresponding to position 192 of SEQ IDNO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 268 of SEQID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the Tcorresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ IDNO:7 is replaced with A; the K corresponding to position 313 of SEQ IDNO:2 or position 294 of SEQ ID NO:7 is replaced with E; the Ncorresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ IDNO:7 is replaced with A; the N corresponding to position 315 of SEQ IDNO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F or A; the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the Kcorresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ IDNO:7 is replaced with A; the R corresponding to position 368 of SEQ IDNO:2 or position 349 of SEQ ID NO:7 is replaced with A; the Fcorresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ IDNO:7 is replaced with A; the K corresponding to position 371 of SEQ IDNO:2 or position 352 of SEQ ID NO:7 is replaced with A; the Lcorresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ IDNO:7 is replaced with A; and the L corresponding to position 373 of SEQID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.

More preferably, SEQ ID NO:2 is modified by the following changes or SEQID NO:7 is modified by the following changes: the Y corresponding toposition 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 193 of SEQ ID NO:2 or position 174 ofSEQ ID NO:7 is replaced with F; the Y corresponding to position 270 ofSEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

Most preferably, SEQ ID NO:2 is modified by the following changes or SEQID NO:7 is modified by the following changes: the Y corresponding toposition 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 193 of SEQ ID NO:2 or position174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 316 of SEQ ID NO:2 or position 297 ofSEQ ID NO:7 is replaced with F; and the Y corresponding to position 362of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

Nucleic Acids

An additional embodiment of the invention is an isolated nucleic acidmolecule segment comprising a structural nucleic acid sequence whichencodes a deallergenized patatin protein.

The structural nucleic acid sequence can generally encode anydeallergenized patatin protein. The structural nucleic acid sequencepreferably encodes a deallergenized patatin protein comprising SEQ IDNO:2 modified in one or more of the following regions, or SEQ ID NO:7modified in one or more of the following regions. The single or multipleamino acid modifications reduce the binding of the modified proteinrelative to the binding of the corresponding unmodified protein. Theregions for modification include amino acid positions 104-113 of SEQ IDNO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ IDNO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). Thepossible amino acid modifications include replacing an amino acid withA, E, F, P, or S. The modifications replace one or more amino acids inthe identified regions, without increasing or decreasing the totalnumber of amino acids in the protein.

Alternatively, the structural nucleic acid sequence encodes SEQ ID NO:2modified by one or more of the following changes or encoding SEQ ID NO:7modified by one or more of the following changes: the Y corresponding toposition 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replacedwith F or A; the I corresponding to position 113 of SEQ ID NO:2 orposition 94 of SEQ ID NO:7 is replaced with A; the Y corresponding toposition 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replacedwith F or A; the K corresponding to position 137 of SEQ ID NO:2 orposition 118 of SEQ ID NO:7 is replaced with A; the S corresponding toposition 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replacedwith A; the Y corresponding to position 185 of SEQ ID NO:2 or position166 of SEQ ID NO:7 is replaced with F or A; the A corresponding toposition 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replacedwith S; the T corresponding to position 192 of SEQ ID NO:2 or position173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding toposition 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replacedwith F or A; the K corresponding to position 268 of SEQ ID NO:2 orposition 249 of SEQ ID NO:7 is replaced with A or E; the T correspondingto position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 isreplaced with A; the Y corresponding to position 270 of SEQ ID NO:2 orposition 251 of SEQ ID NO:7 is replaced with F or A; the K correspondingto position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 isreplaced with A; the K corresponding to position 313 of SEQ ID NO:2 orposition 294 of SEQ ID NO:7 is replaced with E; the N corresponding toposition 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replacedwith A; the N corresponding to position 315 of SEQ ID NO:2 or position296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F orA; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 ofSEQ ID NO:7 is replaced with F; the K corresponding to position 367 ofSEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the Rcorresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ IDNO:7 is replaced with A; the F corresponding to position 369 of SEQ IDNO:2 or position 350 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ IDNO:7 is replaced with A; the L corresponding to position 372 of SEQ IDNO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the Lcorresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ IDNO:7 is replaced with A.

More preferably, the structural nucleic acid sequence encodes SEQ IDNO:2 modified by the following changes or SEQ ID NO:7 modified by thefollowing changes: the Y corresponding to position 106 of SEQ ID NO:2 orposition 87 of SEQ ID NO:7 is replaced with F; the Y corresponding toposition 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 185 of SEQ ID NO:2 or position166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 270 of SEQ ID NO:2 or position 251 ofSEQ ID NO:7 is replaced with F; the Y corresponding to position 316 ofSEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F.

Most preferably, the structural nucleic acid sequence encodes SEQ IDNO:2 modified by the following changes or SEQ ID NO:7 modified by thefollowing changes: the Y corresponding to position 185 of SEQ ID NO:2 orposition 166 of SEQ ID NO:7 is replaced with F; the Y corresponding toposition 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 270 of SEQ ID NO:2 or position251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F;and the Y corresponding to position 362 of SEQ ID NO:2 or position 343of SEQ ID NO:7 is replaced with F.

Recombinant Vectors

An additional embodiment is directed towards recombinant vectorscomprising a structural nucleic acid sequence which encodes adeallergenized patatin protein. The recombinant vector comprisesoperatively linked in the 5′ to 3′ orientation: a promoter that directstranscription of a structural nucleic acid sequence; a structuralnucleic acid sequence, and a 3′ transcription terminator.

The structural nucleic acid sequence can encode SEQ ID NO:2 modified inone or more of the following regions, or SEQ ID NO:7 modified in one ormore of the following regions. The single or multiple amino acidmodifications reduce the binding of the modified protein relative to thebinding of the corresponding unmodified protein. The regions formodification include amino acid positions 104-113 of SEQ ID NO:2 (85-94of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7),184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2(245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ IDNO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possibleamino acid modifications include replacing an amino acid with A, E, F,P, or S. The modifications replace one or more amino acids in theidentified regions, without increasing or decreasing the total number ofamino acids in the protein.

Alternatively, the recombinant vector comprises operatively linked inthe 5′ to 3′ orientation: a promoter that directs transcription of astructural nucleic acid sequence; a structural nucleic acid sequenceencoding SEQ ID NO:2 modified by one or more of the following changes orencoding SEQ ID NO:7 modified by one or more of the following changes:the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQID NO:7 is replaced with F or A; the I corresponding to position 113 ofSEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 137 of SEQID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the Scorresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 185 of SEQ IDNO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the Acorresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ IDNO:7 is replaced with S; the T corresponding to position 192 of SEQ IDNO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 268 of SEQID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the Tcorresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ IDNO:7 is replaced with A; the K corresponding to position 313 of SEQ IDNO:2 or position 294 of SEQ ID NO:7 is replaced with E; the Ncorresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ IDNO:7 is replaced with A; the N corresponding to position 315 of SEQ IDNO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F or A; the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the Kcorresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ IDNO:7 is replaced with A; the R corresponding to position 368 of SEQ IDNO:2 or position 349 of SEQ ID NO:7 is replaced with A; the Fcorresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ IDNO:7 is replaced with A; the K corresponding to position 371 of SEQ IDNO:2 or position 352 of SEQ ID NO:7 is replaced with A; the Lcorresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ IDNO:7 is replaced with A; and the L corresponding to position 373 of SEQID NO:2 or position 354 of SEQ ID NO:7 is replaced with A; and a 3′transcription terminator.

More preferably, the vector comprises a structural nucleic acid sequenceencoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7modified by the following changes: the Y corresponding to position 106of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 185 of SEQ IDNO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

Most preferably, the vector comprises a structural nucleic acid sequenceencoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7modified by the following changes: the Y corresponding to position 185of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

Recombinant Host Cells

A further embodiment of the invention is directed towards recombinanthost cells comprising a structural nucleic acid sequence encoding adeallergenized patatin protein. The recombinant host cell preferablyproduces a deallergenized patatin protein. More preferably, therecombinant host cell produces a deallergenized patatin protein in aconcentration sufficient to inhibit growth or to kill an insect whichingests the recombinant host cell. The recombinant host cell cangenerally comprise any structural nucleic acid sequence encoding adeallergenized patatin protein.

The recombinant host cell can comprise a structural nucleic acidsequence encoding SEQ ID NO:2 modified in one or more of the followingregions, or SEQ ID NO:7 modified in one or more of the followingregions. The single or multiple amino acid modifications reduce thebinding of the modified protein relative to the binding of thecorresponding unmodified protein. The regions for modification includeamino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7),128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2(165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ IDNO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 ofSEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acidmodifications include replacing an amino acid with A, E, F, P, or S. Themodifications replace one or more amino acids in the identified regions,without increasing or decreasing the total number of amino acids in theprotein.

Alternatively, the recombinant host cell comprises a structural nucleicacid sequence encoding SEQ ID NO:2 modified by one or more of thefollowing changes or encoding SEQ ID NO:7 modified by one or more of thefollowing changes: the Y corresponding to position 106 of SEQ ID NO:2 orposition 87 of SEQ ID NO:7 is replaced with F or A; the I correspondingto position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replacedwith A; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F or A; the K corresponding toposition 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replacedwith A; the S corresponding to position 184 of SEQ ID NO:2 or position165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F orA; the A corresponding to position 188 of SEQ ID NO:2 or position 169 ofSEQ ID NO:7 is replaced with S; the T corresponding to position 192 ofSEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; theY corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 268 of SEQID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the Tcorresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ IDNO:7 is replaced with A; the K corresponding to position 313 of SEQ IDNO:2 or position 294 of SEQ ID NO:7 is replaced with E; the Ncorresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ IDNO:7 is replaced with A; the N corresponding to position 315 of SEQ IDNO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F or A; the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the Kcorresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ IDNO:7 is replaced with A; the R corresponding to position 368 of SEQ IDNO:2 or position 349 of SEQ ID NO:7 is replaced with A; the Fcorresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ IDNO:7 is replaced with A; the K corresponding to position 371 of SEQ IDNO:2 or position 352 of SEQ ID NO:7 is replaced with A; the Lcorresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ IDNO:7 is replaced with A; and the L corresponding to position 373 of SEQID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.

More preferably, the recombinant host cell comprises a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by the followingchanges or SEQ ID NO:7 modified by the following changes: the Ycorresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 129 of SEQ IDNO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 193 of SEQ IDNO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F.

Most preferably, the recombinant host cell comprises a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by the followingchanges or SEQ ID NO:7 modified by the following changes: the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 193 of SEQ IDNO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F.

The recombinant host cell can generally be any type of host cell, andpreferably is a bacterial, fungal, or plant cell. The bacterial cell ispreferably an Escherichia coli bacterial cell. The fungal cell ispreferably a Saccharomyces cerevisiae, Schizosaccharomyces pombe, orPichia pastoris fungal cell. The plant cell can be a monocot, dicot, orconifer plant cell. The plant cell is preferably an alfalfa, banana,canola, corn, cotton, cucumber, peanut, potato, rice, soybean,sunflower, sweet potato, tobacco, tomato, or wheat plant cell. Therecombinant host cell preferably further comprises operatively linked tothe structural nucleic acid sequence a promoter that directstranscription of the structural nucleic acid sequence. The recombinanthost cell preferably further comprises operatively linked to thestructural nucleic acid sequence a 3′ transcription terminator and apolyadenylation site.

Recombinant Plants

An additional embodiment of the invention is a recombinant plantcomprising a structural nucleic acid sequence encoding a deallergenizedpatatin protein. The recombinant plant preferably produces adeallergenized patatin protein. More preferably, the recombinant plantproduces a deallergenized patatin protein in a concentration sufficientto inhibit growth or to kill an insect which ingests plant tissue fromthe recombinant plant.

The recombinant plant can comprise a structural nucleic acid sequenceencoding SEQ ID NO:2 modified in one or more of the following regions,or SEQ ID NO:7 modified in one or more of the following regions. Thesingle or multiple amino acid modifications reduce the binding of themodified protein relative to the binding of the corresponding unmodifiedprotein. The regions for modification include amino acid positions104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2(109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ IDNO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 ofSEQ ID NO:7). The possible amino acid modifications include replacing anamino acid with A, E, F, P, or S. The modifications replace one or moreamino acids in the identified regions, without increasing or decreasingthe total number of amino acids in the protein.

Alternatively, the recombinant plant can comprise a structural nucleicacid sequence encoding SEQ ID NO:2 modified by one or more of thefollowing changes or encoding SEQ ID NO:7 modified by one or more of thefollowing changes: the Y corresponding to position 106 of SEQ ID NO:2 orposition 87 of SEQ ID NO:7 is replaced with F or A; the I correspondingto position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replacedwith A; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F or A; the K corresponding toposition 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replacedwith A; the S corresponding to position 184 of SEQ ID NO:2 or position165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F orA; the A corresponding to position 188 of SEQ ID NO:2 or position 169 ofSEQ ID NO:7 is replaced with S; the T corresponding to position 192 ofSEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; theY corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 268 of SEQID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the Tcorresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ IDNO:7 is replaced with A; the K corresponding to position 313 of SEQ IDNO:2 or position 294 of SEQ ID NO:7 is replaced with E; the Ncorresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ IDNO:7 is replaced with A; the N corresponding to position 315 of SEQ IDNO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F or A; the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the Kcorresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ IDNO:7 is replaced with A; the R corresponding to position 368 of SEQ IDNO:2 or position 349 of SEQ ID NO:7 is replaced with A; the Fcorresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ IDNO:7 is replaced with A; the K corresponding to position 371 of SEQ IDNO:2 or position 352 of SEQ ID NO:7 is replaced with A; the Lcorresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ IDNO:7 is replaced with A; and the L corresponding to position 373 of SEQID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.

More preferably, the recombinant plant comprises a structural nucleicacid sequence encoding SEQ ID NO:2 modified by the following changes orSEQ ID NO:7 modified by the following changes: the Y corresponding toposition 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 193 of SEQ ID NO:2 or position 174 ofSEQ ID NO:7 is replaced with F; the Y corresponding to position 270 ofSEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

Most preferably, the recombinant plant comprises a structural nucleicacid sequence encoding SEQ ID NO:2 modified by the following changes orSEQ ID NO:7 modified by the following changes: the Y corresponding toposition 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 193 of SEQ ID NO:2 or position174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 316 of SEQ ID NO:2 or position 297 ofSEQ ID NO:7 is replaced with F; and the Y corresponding to position 362of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

The recombinant plant can generally be any type of plant. The plant canbe a monocot, dicot, or conifer plant. The plant is preferably analfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice,soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.

The recombinant plant preferably further comprises operatively linked tothe structural nucleic acid sequence a promoter that directstranscription of the structural nucleic acid sequence. The recombinantplant preferably further comprises operatively linked to the structuralnucleic acid sequence a 3′ transcription terminator and apolyadenylation site.

Methods of Preparation

Embodiments of the invention are further directed towards methods ofpreparing recombinant host cells and recombinant plants useful for theproduction of deallergenized patatin proteins.

A method of preparing a recombinant host cell useful for the productionof deallergenized patatin proteins can comprise selecting a host cell;transforming the host cell with a recombinant vector; and obtainingrecombinant host cells.

The recombinant vector comprises a structural nucleic acid sequenceencoding SEQ ID NO:2 modified in one or more of the following regions,or SEQ ID NO:7 modified in one or more of the following regions. Thesingle or multiple amino acid modifications reduce the binding of themodified protein relative to the binding of the corresponding unmodifiedprotein. The regions for modification include amino acid positions104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2(109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ IDNO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 ofSEQ ID NO:7). The possible amino acid modifications include replacing anamino acid with A, E, F, P, or S. The modifications replace one or moreamino acids in the identified regions, without increasing or decreasingthe total number of amino acids in the protein.

Alternatively, the recombinant vector comprises a structural nucleicacid sequence encoding SEQ ID NO:2 modified by one or more of thefollowing changes or encoding SEQ ID NO:7 modified by one or more of thefollowing changes: the Y corresponding to position 106 of SEQ ID NO:2 orposition 87 of SEQ ID NO:7 is replaced with F or A; the I correspondingto position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replacedwith A; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F or A; the K corresponding toposition 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replacedwith A; the S corresponding to position 184 of SEQ ID NO:2 or position165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F orA; the A corresponding to position 188 of SEQ ID NO:2 or position 169 ofSEQ ID NO:7 is replaced with S; the T corresponding to position 192 ofSEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; theY corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 268 of SEQID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the Tcorresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ IDNO:7 is replaced with A; the K corresponding to position 313 of SEQ IDNO:2 or position 294 of SEQ ID NO:7 is replaced with E; the Ncorresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ IDNO:7 is replaced with A; the N corresponding to position 315 of SEQ IDNO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F or A; the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the Kcorresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ IDNO:7 is replaced with A; the R corresponding to position 368 of SEQ IDNO:2 or position 349 of SEQ ID NO:7 is replaced with A; the Fcorresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ IDNO:7 is replaced with A; the K corresponding to position 371 of SEQ IDNO:2 or position 352 of SEQ ID NO:7 is replaced with A; the Lcorresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ IDNO:7 is replaced with A; and the L corresponding to position 373 of SEQID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.

More preferably, the vector comprises a structural nucleic acid sequenceencoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7modified by the following changes: the Y corresponding to position 106of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 185 of SEQ IDNO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

Most preferably, the vector comprises a structural nucleic acid sequenceencoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7modified by the following changes: the Y corresponding to position 185of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

The method can generally be used to prepare any type of recombinant hostcell. Preferably, the method can be used to prepare a recombinantbacterial cell, a recombinant fungal cell, or a recombinant plant cell.The bacterial cell is preferably an Escherichia coli bacterial cell. Thefungal cell is preferably a Saccharomyces cerevisiae,Schizosaccharomyces pombe, or Pichia pastoris fungal cell. The plantcell can be a monocot, dicot, or conifer plant cell. The plant cell ispreferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut,potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, orwheat plant cell.

An additional embodiment is directed towards methods for the preparationof recombinant plants useful for the production of deallergenizedpatatin proteins. The method can comprise selecting a host plant cell;transforming the host plant cell with a recombinant vector; obtainingrecombinant host cells; and regenerating a recombinant plant from therecombinant host plant cells.

The recombinant vector comprises a structural nucleic acid sequenceencoding SEQ ID NO:2 modified in one or more of the following regions,or SEQ ID NO:7 modified in one or more of the following regions. Thesingle or multiple amino acid modifications reduce the binding of themodified protein relative to the binding of the corresponding unmodifiedprotein. The regions for modification include amino acid positions104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2(109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ IDNO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 ofSEQ ID NO:7). The possible amino acid modifications include replacing anamino acid with A, E, F, P, or S. The modifications replace one or moreamino acids in the identified regions, without increasing or decreasingthe total number of amino acids in the protein.

Alternatively, the recombinant vector comprises a structural nucleicacid sequence encoding SEQ ID NO:2 modified by one or more of thefollowing changes or encoding SEQ ID NO:7 modified by one or more of thefollowing changes: the Y corresponding to position 106 of SEQ ID NO:2 orposition 87 of SEQ ID NO:7 is replaced with F or A; the I correspondingto position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replacedwith A; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F or A; the K corresponding toposition 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replacedwith A; the S corresponding to position 184 of SEQ ID NO:2 or position165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F orA; the A corresponding to position 188 of SEQ ID NO:2 or position 169 ofSEQ ID NO:7 is replaced with S; the T corresponding to position 192 ofSEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; theY corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 268 of SEQID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the Tcorresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ IDNO:7 is replaced with A; the K corresponding to position 313 of SEQ IDNO:2 or position 294 of SEQ ID NO:7 is replaced with E; the Ncorresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ IDNO:7 is replaced with A; the N corresponding to position 315 of SEQ IDNO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F or A; the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the Kcorresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ IDNO:7 is replaced with A; the R corresponding to position 368 of SEQ IDNO:2 or position 349 of SEQ ID NO:7 is replaced with A; the Fcorresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ IDNO:7 is replaced with A; the K corresponding to position 371 of SEQ IDNO:2 or position 352 of SEQ ID NO:7 is replaced with A; the Lcorresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ IDNO:7 is replaced with A; and the L corresponding to position 373 of SEQID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.

More preferably, the vector comprises a structural nucleic acid sequenceencoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7modified by the following changes: the Y corresponding to position 106of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 185 of SEQ IDNO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

Most preferably, the vector comprises a structural nucleic acid sequenceencoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7modified by the following changes: the Y corresponding to position 185of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

The recombinant plant can generally be any type of plant. The plant canbe a monocot, dicot, or conifer plant. The plant is preferably analfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice,soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.

Deallergenized patatin proteins can be prepared by isolating thedeallergenized patatin protein from any one of the above described hostcells or plants.

Deglycosylation

The examples herein provide evidence that glycosylation of cancontribute to the allergenicity of a protein. Accordingly, rationalsubstitution of amino acid residues likely to be the targets ofglycosylation within a subject allergen protein may reduce or eliminatethe allergenic properties of the protein without adversely affecting theenzymatic, insecticidal, antifungal or other functional properties ofthe protein.

Glycosylation commonly occurs as either N-linked or O-linked forms.N-linked glycosylation usually occurs at the motif Asn-Xaa-Ser/Thr,where Xaa is any amino acid except Pro (Kasturi, L. et al., Biochem J.323: 415-519, 1997; Melquist, J. L. et al., Biochemistry 37: 6833-6837,1998). O-linked glycosylation occurs between the hydroxyl group ofserine or threonine and an amino sugar.

Site directed mutagenesis of selected asparagine, serine, or threoninemay be used to reduce or eliminate the glycosylation of patatinproteins. A search of SEQ ID NO:2 for the Asn-Xaa-Ser/Thr motif revealsone occurrence at amino acid positions 202-204. Mutagenization of thenucleic acid sequence encoding this region may result in a reducedallergenicity of the encoded protein.

In order to test this conceptual approach to reducing allergenicity ofpatatin proteins, two sets of experiments were performed: a) productionof patatin proteins in Escherichia coli, which do not glycosylateproteins; and b) production of patatin proteins with an N202Q sitedirected mutation.

Antibodies obtained from patients HS-07 and G15-MON (not potatoallergic) did not show specific binding to wild type patatin, patatinproduced in E. coli, or the N202Q variant. Antibodies obtained frompatient HS-01 (potato allergic) bound to wild type patatin, but not topatatin produced in E. coli or the N202Q variant. Antibodies obtainedfrom patient HS-02 (potato allergic) bound strongly to wild typepatatin, but extremely weakly to patatin produced in E. coli, andbinding to the N202Q variant resembled vector controls. Antibodiesobtained from patient HS-03 (potato allergic) bound to wild typepatatin, but not to patatin produced in E. coli or the N202Q variant.Antibodies obtained from patient HS-05 (potato allergic) bound to wildtype patatin, but very weakly to patatin produced in E. coli and theN202Q variant. Antibodies obtained from patient HS-06 (potato allergic)strongly bound wild type patatin, the N202Q variant, and to patatinproduced in E. coli. These results strongly suggest that glycosylationis at least partially responsible for the antigenic properties ofpatatin proteins, and that site directed mutagenesis may be used toreduce or eliminate specific antibody binding. Mutagenesis at position202 of SEQ ID NO:2 may be useful for reducing or eliminating specificantibody binding.

Permuteins

The positions of the internal breakpoints described in the followingExamples are found on the protein surface, and are distributedthroughout the linear sequence without any obvious bias towards the endsor the middle. Breakpoints occurring below the protein surface canadditionally be selected. The rearranged two subunits can be joined by apeptide linker. A preferred embodiment involves the linking of theN-terminal and C-terminal subunits by a three amino acid linker,although linkers of various sizes can be used. Additionally, theN-terminal and C-terminal subunits can be joined lacking a linkersequence. Furthermore, a portion of the C-terminal subunit can bedeleted and the connection made from the truncated C-terminal subunit tothe original N-terminal subunit and vice versa as previously described(Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980-11984,1993; Viguera, et al., Mol. Biol., 247: 670-681, 1995; Protasova, etal., Prot. Eng., 7: 1373-1377, 1994).

The novel insecticidal proteins of the present invention can berepresented by the formula:X¹-(L)_(a)-X²

wherein;

-   -   a is 0 or 1, and if a is 0, then the permutein does not contain        a linker sequence;    -   X¹ is a polypeptide sequence corresponding to amino acids n+1        through J;    -   X² is a polypeptide corresponding to amino acids 1 through n;    -   n is an integer ranging from 1 to J-1;    -   J is an integer greater than n+1; and    -   L is a linker.

In the formula above, the constituent amino acid residues of the novelinsecticidal protein are numbered sequentially 1 through J from theoriginal amino terminus to the original carboxyl terminus. A pair ofadjacent amino acids within this protein can be numbered n and n+1respectively where n is an integer ranging from 1 to J-1. The residuen+1 becomes the new N-terminus of the novel insecticidal protein and theresidue n becomes the new C-terminus of the novel insecticidal protein.

For example, a parent protein sequence consisting of 120 amino acids canbe selected as a starting point for designing a permutein (J=120). Ifthe breakpoint is selected as being between position 40 and position 41,then n=40. If a linker is selected to join the two subunits, theresulting permutein will have the formula: (amino acids 41-120)-L-(aminoacids 1-40). If a linker was not used, the resulting permutein will havethe formula: (amino acids 41-120)-(amino acids 1-40).

The length of the amino acid sequence of the linker can be selectedempirically, by using structural information, or by using a combinationof the two approaches. When no structural information is available, asmall series of linkers can be made whose length can span a range of 0to 50 Å and whose sequence is chosen in order to be substantiallyconsistent with surface exposure (Hopp and Woods, Mol. Immunol., 20:483-489, 1983; Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982;Lee and Richards, J. Mol. Biol., 55: 379-400, 1971) and the ability toadopt a conformation which does not significantly affect the overallconfiguration of the protein (Karplus and Schulz, Naturwissenschaften,72: 212-213, 1985). Assuming an average length of 2.0 to 3.8 Å perresidue, this would mean the length to test would be between about 0 toabout 30 residues, with 0 to about 15 residues being the preferredrange. Accordingly, there are many such sequences that vary in length orcomposition that can serve as linkers with the primary considerationbeing that they be neither excessively long nor excessively short(Sandhu, et al., Critical Rev. Biotech., 12: 437-467, 1992). If thelinker is too long, entropy effects may destabilize thethree-dimensional fold and may affect protein folding. If the linker istoo short, it may destabilize the molecule due to torsional or stericstrain.

Use of the distance between the chain ends, defined as the distancebetween the C-alpha carbons, can be used to define the length of thesequence to be used, or at least to limit the number of possibilitiesthat can be tested in an empirical selection of linkers. Using thecalculated length as a guide, linkers with a range of number of residues(calculated using 2 to 3.8 Å per residue) can be selected. These linkerscan be composed of the original sequence, shortened or lengthened asnecessary, and when lengthened the additional residues can be chosen tobe flexible and hydrophilic as described above; or optionally theoriginal sequence can be substituted for using a series of linkers, oneexample being Gly-Pro-Gly (SEQ ID NO:277); or optionally a combinationof the original sequence and new sequence having the appropriate totallength can be used. An alternative short, flexible linker sequence isGly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276).

Selection of Permutein Breakpoints

Sequences of novel patatin analogs capable of folding to biologicallyactive molecules can be prepared by appropriate selection of thebeginning (amino terminus) and ending (carboxyl terminus) positions fromwithin the original polypeptide chain while optionally using a linkersequence as described above. Amino and carboxyl termini can be selectedfrom within a common stretch of sequence, referred to as a breakpointregion, using the guidelines described below. A novel amino acidsequence is thus generated by selecting amino and carboxyl termini fromwithin the same breakpoint region. In many cases, the selection of thenew termini will be such that the original position of the carboxylterminus immediately preceded that of the amino terminus. However,selections of termini anywhere within the region may result in afunctional protein, and that these will effectively lead to eitherdeletions or additions to the amino or carboxyl portions of the newsequence.

The primary amino acid sequence of a protein dictates folding to thethree-dimensional structure beneficial for expression of its biologicalfunction. It is possible to obtain and interpret three-dimensionalstructural information using x-ray diffraction of single proteincrystals or nuclear magnetic resonance spectroscopy of proteinsolutions. Examples of structural information that are relevant to theidentification of breakpoint regions include the location and type ofprotein secondary structure (alpha and 3-10 helices, parallel andanti-parallel beta sheets, chain reversals and turns, and loops (Kabschand Sander, Biopolymers, 22: 2577-2637, 1983), the degree of solventexposure of amino acid residues, the extent and type of interactions ofresidues with one another (Chothia, C., Ann. Rev. Biochem., 53: 537-572,1984), and the static and dynamic distribution of conformations alongthe polypeptide chain (Alber and Mathews, Methods Enzymol., 154:511-533, 1987). In some cases additional information is known aboutsolvent exposure of residues, one example is a site ofpost-translational attachment of carbohydrate which is necessarily onthe surface of the protein. When experimental structural information isnot available, or when it is not feasible to obtain the information,methods are available to analyze the primary amino acid sequence inorder to make predictions of protein secondary and tertiary structure,solvent accessibility and the occurrence of turns and loops (Fasman, G.,Ed. Plenum, New York, 1989; Robson, B. and Gamier, J. Nature 361: 506,1993).

Biochemical methods can be applicable for empirically determiningsurface exposure when direct structural methods are not feasible; forexample, using the identification of sites of chain scission followinglimited proteolysis in order to infer surface exposure (Gentile, F. andSalvatore, G., Eur. J. Biochem., 218: 603-621, 1993). Thus, using eitherthe experimentally derived structural information or predictive methods(Srinivasan, R. and Rose, G. D. Proteins, 22: 81-99, 1995), the parentalamino acid sequence can be analyzed to classify regions according towhether or not they are integral to the maintenance of secondary andtertiary structure. The sequences within regions that are known to beinvolved in periodic secondary structure (alpha and 3-10 helices,parallel and anti-parallel beta sheets) are regions that should beavoided. Similarly, regions of amino acid sequence that are observed orpredicted to have a low degree of solvent exposure are more likely to bepart of the so-called hydrophobic core of the protein and should also beavoided for selection of amino and carboxyl termini. Regions that areknown or predicted to be in surface turns or loops, and especially thoseregions that are known not to be required for biological activity, canbe preferred sites for new amino and carboxyl termini. Stretches ofamino acid sequence that are preferred based on the above criteria canbe selected as breakpoint regions.

An embodiment of the invention is directed towards patatin permuteinproteins. The permutein proteins preferably maintain esterase activityand insecticidal properties. The permutein proteins preferably are lessallergenic than the wild type patatin protein to individuals or animalsallergic to potatoes. This can be assayed by the binding of antibodiesto the wild type patatin and patatin permutein proteins.

The permutein proteins can optionally contain a linker sequence. Thelinker can generally be any amino acid sequence, preferably isGly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276) or Gly-Pro-Gly (SEQ IDNO:277), and more preferably is Gly-Pro-Gly (SEQ ID NO:277). Specificpermutein proteins comprise: (amino acids 247-386 of SEQ IDNO:2)-linker-(amino acids 24-246 of SEQ ID NO:2), (amino acids 269-386of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2), SEQ IDNO:247, and SEQ ID NO:259.

Embodiments of the invention also include isolated nucleic acid moleculesegments comprising a structural nucleic acid sequence encoding apatatin permutein protein. The encoded permutein protein can generallybe any permutein protein, and preferably comprises (amino acids 247-386of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2), (amino acids269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2), SEQID NO:247, or SEQ ID NO:259. The linker can generally be any amino acidsequence, preferably is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276) orGly-Pro-Gly (SEQ ID NO:277), and more preferably is Gly-Pro-Gly (SEQ IDNO:277). Alternatively, the encoded patatin permutein protein can lack alinker sequence. The structural nucleic acid sequence is preferably SEQID NO:246 or SEQ ID NO:258.

An embodiment of the invention is directed towards recombinant vectorswhich encode a patatin permutein protein. The vector can compriseoperatively linked in the 5′ to 3′ orientation: a promoter that directstranscription of a structural nucleic acid sequence; a structuralnucleic acid sequence encoding a protein selected from the groupconsisting of: (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids24-246 of SEQ ID NO:2); and (amino acids 269-386 of SEQ IDNO:2)-linker-(amino acids 24-268 of SEQ ID NO:2); and a 3′ transcriptionterminator. The linker can comprise Gly-Pro-Gly (SEQ ID NO:277) orGly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively, the encodedpatatin permutein protein can lack a linker sequence. The structuralnucleic acid sequence can preferably be SEQ ID NO:246 or SEQ ID NO:258,and preferably encodes SEQ ID NO:247 or SEQ ID NO:259.

An additional embodiment of the invention is directed towardsrecombinant host cells useful for the production of a patatin permuteinprotein. The recombinant host cell preferably produces a patatinpermutein protein. More preferably, the recombinant host cell produces apatatin permutein protein in a concentration sufficient to inhibitgrowth or to kill an insect which ingests the recombinant host cell. Therecombinant host cell can comprise a structural nucleic acid sequenceencoding a protein selected from the group consisting of: (amino acids247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2); and(amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQID NO:2). The linker can generally be any amino acid sequence, andpreferably is Gly-Pro-Gly (SEQ ID NO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly(SEQ ID NO:276). Alternatively, the encoded patatin permutein proteincan lack a linker sequence. The structural nucleic acid sequence ispreferably SEQ ID NO:246 or SEQ ID NO:258, and preferably encodes SEQ IDNO:247 or SEQ ID NO:259. The structural nucleic acid sequence can beoperatively linked to a promoter sequence that directs transcription ofthe structural nucleic acid sequence, a 3′ transcription terminator, anda 3′ polyadenylation signal sequence. The recombinant host cell cangenerally be any type of host cell, and preferably is a bacterial,fungal, or plant host cell. The bacterial cell is preferably anEscherichia coli bacterial cell. The fungal cell is preferably aSaccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastorisfungal cell. The plant cell can be a monocot, dicot, or conifer plantcell. The plant cell is preferably an alfalfa, banana, canola, corn,cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweetpotato, tobacco, tomato, or wheat plant cell.

An additional embodiment of the invention is directed towardsrecombinant plants which are useful for the production of patatinpermutein proteins. The recombinant plant preferably produces a patatinpermutein protein. More preferably, the recombinant plant produces apatatin permutein protein in a concentration sufficient to inhibitgrowth or to kill an insect which ingests tissue from the recombinantplant. The recombinant plant can comprise a structural nucleic acidsequence encoding a protein selected from the group consisting of:(amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQID NO:2); and (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids24-268 of SEQ ID NO:2). The linker can comprise Gly-Pro-Gly (SEQ IDNO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively,the encoded protein can lack a linker sequence. The structural nucleicacid sequence is preferably SEQ ID NO:246 or SEQ ID NO:258, andpreferably encodes SEQ ID NO:247 or SEQ ID NO:259. The structuralnucleic acid sequence can be operatively linked to a promoter sequencethat directs transcription of the structural nucleic acid sequence, a 3′transcription terminator, and a 3′ polyadenylation signal sequence. Therecombinant plant can generally be any type of plant, and preferably isan alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato,rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.

Permutein proteins can be prepared by isolating the permutein proteinfrom any one of the above described host cells or plants.

Immunotherapy for Potato Allergy

Immunotherapy for food allergy has been largely unsuccessful due to thelack of appropriate therapeutic reagents (Sampson, H. A., J. AllergyClin. Immunol., 90(2): 151-152, 1992). Immunotherapy has typicallyinvolved the administration (orally or by subcutaneous injections) ofincreasing doses of crude protein extracts of the offending allergenicentities which usually contain variable mixes of many different proteins(Scheiner, O., Wien Klin Wochenschr., 105(22): 653-658, 1993). Whilethere are reports of highly successful clinical applications ofimmunotherapy for food allergens (Romano, P. C., et al., Allergol.Immunopathol. (Madr), 12(4): 275-281, 1984), those reports are rare andthe clinical literature in general recommends avoidance far morestrongly than therapy (Gay, G., Allerg. Immunol. (Paris), 29(6):169-170, 1997). One of the primary reasons for the failure of manyclinical attempts to induce tolerance to allergens in general and foodallergens in particular relates to anecdotal comments by numerousallergists, that patients don't tolerate the doses of allergen requiredto achieve tolerance. Animal studies examining the relationship ofantigen dose and the induction of tolerance have demonstrated a strongpositive correlation (Chen, Y., et al., Proc. Natl. Acad. Sci., U.S.A.,93: 388-391, 1996; Tokai, T., et al., Nat. Biotechnol., 15(8): 754-758,1997). Due to the very real possibility of inducing an anaphylacticreaction in patients with native allergen, most clinical therapists arequite hesitant to use high doses therapeutically and are thereforecompromising the likelihood of successful therapy.

In recent reports, recombinant technology has been used to reduce theallergenic potential of a major allergen without modifying the T cellepitopes, and allowing higher doses of protein to be used in therapy(Tokai, T., et al., Nat. Biotechnol., 15(8): 754-758, 1997). Inaddition, a lack of understanding about the appropriate route ofadministration, the uncertainty of mechanisms responsible for inductionof allergy and the uncertainty of mechanisms by which immunotherapysuppresses or blocks the T cell-IgE-eosinophil/mast cell cycle havecontributed to the large number of equivocal studies and clinicaltrials. Recent studies in animal models dealing with mechanisms, routesof administration, adjuvants and vaccine formulations have increased thelikelihood that immunotherapy for allergies, including food allergies,will become a reproducibly successful clinical treatment when theappropriate therapeutic reagents are available (Sampson, H. A. andBurks, A. W., Annu. Rev. Nutr., 16: 161-177, 1996; Kaminogawa, S.,Biosci. Biotechnol. Biochem., 60(11): 1749-1756, 1996; Chapman, M. D.,et al., Allergy, 52: 374-379, 1997; Barbeau, W. E., Adv. Exp. Med.Biol., 415: 183-193, 1997; Cao, Y, et al., Immunology, 90(1): 46-51,1997; Garside, P. and Mowat, A. M., Crit. Rev. Immunol., 17(2): 119-137,1997; Rothe, M. J. and Grant-Kels, J. M., J. Am. Acad. Dermatol., 35(1):1-13, 1996; Strobel, S., Allergy, 50(20): 18-25, 1995; Kruisbeek, A. M.and Amsen, D., Curr. Opin. Immunol., 8(2): 233-244, 1996; Herz, U., etal., Adv. Exp. Med. Biol., 409: 25-32, 1996; Litwin, A., et al., J.Allergy Clin. Immunol., 100: 30-38, 1997; Vandewalker, M. L., Mo. Med.,94(7): 311, 1997; Marshall, G. D., Jr. and Davis, F., Nat. Biotechnol.,15(8): 718-719, 1997; Van Deusen, M. A., et al., Ann. Allergy AsthmaImmunol., 78: 573-580, 1997; Jacobsen, L., et al., Allergy, 52: 914-920,1997, Scheiner, O. and Kraft, D., Allergy 50(5): 384-391, 1995).

Relative to immunotherapy, the critical aspects of the modified patatingenes described in this patent are that they can be used to synthesizepurified, deallergenized-protein which can be used for patatin (potato)specific immunotherapy, with reduced potential for adverse andpotentially fatal anaphylactic reactions in human or veterinary patientswho have allergies to patatin or potatoes. Various strategies, includingfixing or cross linking allergens, encapsulation of allergen for oraldelivery, the use of small, T-cell epitope peptides and most recently,the use of engineered recombinant proteins, or modified gene vaccinesare being tested in attempts to decrease the potential for anaphylacticreactions while inducing tolerance (Cao, Y., et al., Immunology, 90(1):46-51, 1997; Chapman, M. D., et al., Allergy, 52: 374-379, 1997;Chapman, M. D., et al., Int. Arch. Allergy Immunol., 113(1-3): 102-104,1997; Collins, S. P., et al., Clin. Exp. Allergy, 26(1): 36-42, 1996;Takai, T., et al., Mol. Immunol., 34(3): 255-261, 1997; Takai, T., etal., Nat. Biotechnol., 15(8) 754-758, 1997; Jirapongsananruk, O. andLeung, D. Y. M., Ann. Allergy Asthma Immunol., 79: 5-20, 1997; Litwin,A., et al., J. Allergy Clin. Immunol., 100: 30-38, 1997; Vandewalker, M.L., Mo. Med., 94(7): 311, 1997; Raz, E., et al., Proc. Natl. Acad. Sci.,U.S.A., 93: 5141-5145, 1996; Hoyne, G. F., et al., Clin. Immunol.Immunopathol., 80: S23-30, 1996; Hoyne, G. F., et al., Int. Immunol.,9(8): 1165-1173, 1997; Vrtala, S., et al., J. Clin. Invest., 99(7):1673-1681, 1997; Sato, Y., et al., Science, 273: 352-354, 1996; Lee, D.J., et al., Int. Arch. Allergy Immunol., 113(1-3): 227-230, 1997;Tsitoura, D. C., et al., J. Immunol., 157(5): 2160-2165, 1996; Hsu, C.H., et al., Int. Immunol., 8(9):1405-1411, 1996; Hsu, C. H., et al.,Nat. Med., 2(5): 540-544, 1996).

The instant invention uses an engineered patatin protein, as expressedin any living cell, with or without post-synthesis modifications, forimmunotherapy by the routes of cutaneous or subcutaneous exposure,injection, or by oral, gastro-intestinal, respiratory or nasalapplication, either with, or without the use of specific carriers,vehicles and adjuvants. The direct application of nucleic acid encodingrecombinant patatin as the in vivo (in the patient) expression template(gene) as RNA-, DNA- or gene-vaccines is also the intended use of theengineered genetic materials defined here, coding for patatin, but withmodified IgE binding sites. It is also the intent of this patent tocover the use of these modified genes described here including insertioninto various DNA vectors including adenovirus, retrovirus, pox virus andreplicating or non-replicating eukaryotic expression plasmids (Lee, D.J., et al., Int. Arch. Allergy Immunol., 113(1-3): 227-230, 1997) withvarious promoters and regulatory sequences, which can be inserted intothe patient's somatic cells (dendritic cells, epithelial cells, musclefiber-cells, fibroblasts, etc.) for the purpose of expressing therecombinant gene product to alter the patient's immune response to thepatatin proteins (Lee D. J., et al., Int. Arch. Allergy Immunol.,113(1-3): 227-230, 1997). Potential routes of administration foreseen inthis application include previously described methods of encapsulation,emulsion, receptor or membrane fusion mediated uptake and methods ofdirect permeabilization or insertion of the DNA or corresponding RNAinto the host cells.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES Example 1

Identification of Patatin as an Allergen

Since patatin is commonly obtained from an allergenic source (potato),it was hypothesized that patatins in fact encode an important class ofoffending potato allergens (patatin was reported as allergenic bySeppala, U. et al., J. Allergy Clin. Immunol. 103: 165-171, 1999).Assessment of potential allergens preferably include appropriate invitro testing for IgE binding, in this case with potato allergic sera(Fuchs, R. L. and Astwood, J. D., Food Technology, 50: 83-88, 1996;Astwood, J. D., et al., Monographs in allergy Vol. 32: Highlights infood allergy, pp. 105-120, 1996, Metcalfe, D. D., et al., CriticalReviews in Food Science and Nutrition, 36S: 165-186, 1996). It is therecommendation of a working group organized by the IFBC and the ILSIAllergy and Immunology Institute that proteins encoded by nucleic acidsequences from allergenic sources such as potato (a “less-commonly”allergenic source) should be examined for their ability to react withIgEs of potato-allergic patients using a minimum of five individualpatient sera (Metcalfe, D. D., et al., Critical Reviews in Food Scienceand Nutrition, 36S: 165-186, 1996). Patatin-17 protein was tested forIgE binding using standard in vitro testing with serum taken frompatients with bona fide well defined clinically displayed potato allergyas described below.

Clinical Characterization of Potato Allergic Subjects (Serum Donors)

Patients who suffer from potato allergy were identified at Johns HopkinsClinic (Baltimore, Md.) and were evaluated for potato allergy usingclinical criteria outlined in Table 2.

Serum was obtained from patients with convincing clinical history ofpotato allergy. The convincing history was defined as being one or moreof the following: a) positive potato allergic as evaluated bydouble-blind placebo-control food challenge b) anaphylaix and/orhospitalization due to the consumption of potatoes or c) dramatic skintest results.

TABLE 2 Clinical patient data Flare/Wheal Patient Clinical History (Skinprick test) DBPCFC (potato) HS01 Most recent hospitalization: Oct. 19,1993  7/19, 4/14, 7/17 Not performed AD, A, AR, FH, MFS, IgE = 1397KIAUa/L HS02 Most recent hospitalization: Jun. 1994 20/26 Not performedAD, FH, Latex (+) RAST, MFS, IgE = 7544 K/L HS03 Most recenthospitalization: Jul. 27, 1995  5/13 Yes AD, A, FH, MFS, IgE = N/A HS05Most recent hospitalization May 30, 1995  4/9 Yes AD, A, FH, MFS, IgE =12341 ng/ml HS06 Most recent hospitalization Jun. 13, 1995  5/20, 4/13,5/12 Yes AD, A FH, MFS IgE = N/A HS07 Not potato allergic, allergic toegg, milk, High IgE control serum, not peanuts, seafood. AD, A, AR, FH,MFS allergic to potato. HS08 Non-atopic (normal) Low IgE control serumAD = Atopic dermatitis; FH = Food hypersensitivity; AR = Allergicrhinitis; A = Asthma; MFS = Multiple food sensitivity; N/A = notavailable.

Example 2

Western Blotting of Patatin Proteins

Western blotting experiments were performed using patatin proteinpurified to near homogeneity from corn plants genetically engineered toproduce patatin, patatin producing crude genetically engineered cornleaf extracts, crude potato tuber extracts, and non-transgenic corn leafsamples.

Protein samples were electrophoresed by SDS-PAGE (Laemmli, U. K., Nature227: 680-685, 1970) and were electroblotted onto nitrocellulose. Proteinblots were processed by standard Western blotting (immunoblotting)techniques and were incubated in potato allergic serum diluted 1:5 inPBS buffer for 1 hour. After washing the blots 3 times with PBS, theblots were incubated in biotinylated anti-IgE (Johns Hopkins Hospital,Baltimore, Md.) for 1 hour, followed by a 30 minute incubation inHRP-linked avidin (Promega, New York, N.Y.). IgE-reactive protein bandswere visualized by DAB staining (3,3 diaminobenzidine). The blots weredried and photographed. Individual blots are labeled according topatient serum used. As a control, one blot was incubated in anti-IgEonly.

Patatins were shown to be an allergen of potato by examining thereactivity of purified patatin to sera obtained from patients allergicto potato. Sera from five potato allergic subjects were tested byWestern blotting techniques. All five sera reacted with purified patatinprotein.

Patatin isozymes (SEQ ID NOS:278-282, FIG. 1) were tested for IgEbinding by Western blotting. Isozymes of patatin were cloned into ayeast expression system and purified prior to analysis. The isozymeswere subjected to IgE western blotting as described above with theexception that all five patient sera were pooled. The resulting Westernblot of the yeast-expressed isozymes showed that all five isozymes boundIgE in a manner similar to patatin 17, and that all isozymes of patatintested are also allergens.

Example 3

Western Blotting of Patatin Proteins

Eighty-nine 10-mer peptides were synthesized using the Genosys SPOTssystem, each consecutive 10-mer overlapping by 6 amino acids based onthe amino acid sequence of patatin 17 (SEQ ID NO:2). The peptides wereevaluated for IgE binding with five different potato allergic patientsera using the same incubation procedures as described above. Theresults are summarized graphically in FIG. 2, showing major and minorallergenic epitopes. Interestingly, many of the immunogenic epitopescontain tyrosine. The peptide numbers, sequences, and immunoreactivityis detailed in Table 3.

TABLE 3 Peptide scan of patatin 17 Peptide # (SEQ ID Cumulative NO)Peptide Sequence HS01 HS02 HS03 HS05 HS06 Total  1 (16) QLGEMVTVLS 0.470.33 0.02 0.05 0.06 0.93  2 (17) MVTVLSIDGG 0.53 0.33 0.02 0.07 0.05 1 3 (18) LSIDGGGIRG 0.52 0.38 0.07 0.08 0.09 1.14  4 (19) GGGIRGIIPA 0.530.19 0.06 0.19 0.23 1.2  5 (20) RGIIPATILE 0.46 0.28 0.04 0.09 0.05 0.92 6 (21) PATILEFLEG 0.49 0.31 0.05 0.09 0.07 1.01  7 (22) LEFLEGQLQE 0.360.24 0.04 0.1 0.06 0.8  8 (23) EGQLQEMDNN 0.29 0.19 0.02 0.09 0.05 0.64 9 (24) QEMDNNADAR 0.22 0.13 0.01 0.05 0.04 0.45 10 (25) NNADARLADY 0.210.17 0.03 0.05 0.07 0.53 11 (26) ARLADYFDVI 0.54 0.31 0.16 0.15 0.251.41 12 (27) DYFDVIGGTS 0.61 0.34 0.46 0.06 0.15 1.62 13 (28) VIGGTSTGGL0.63 0.72 0.05 0.15 0.09 1.64 14 (29) TSTGGLLTAM 0.3 0.17 0.03 0.06 0.090.65 15 (30) GLLTAMISTP 0.63 0.41 0.05 0.24 0.12 1.45 16 (31) AMISTPNENN0.34 0.18 0.02 0.07 0.02 0.63 17 (32) TPNENNRPFA 0.46 0.22 0.03 0.190.07 0.97 18 (33) NNRPFAAAKE 0.37 0.21 0.05 0.07 0.06 0.76 19 (34)FAAAKEIVPF 0.52 0.29 0.08 0.11 0.08 1.08 20 (35) KEIVPFYFEH 0.29 0.140.28 0.29 0.23 1.23 21 (36) PFYFEHGPQI 0.65 0.06 1.08 0.51 0.17 2.47 22(37) EHGPQIFNPS 0.34 0.15 0.03 0.05 0.06 0.63 23 (38) QIFNPSGQIL 0.330.29 0.02 0.07 0.07 0.78 24 (39) PSGQILGPKY 0 0 0.02 0 0.05 0.07 25 (40)ILGPKYDGKY 0 0 0.07 0 0.02 0.09 26 (41) KYDGKYLMQV 0.02 0 0.11 0.01 0.040.18 27 (42) KYLMQVLQEK 0.12 0.04 1.08 0.07 0.79 2.1 28 (43) QVLQEKLGET0.46 0.16 0.01 0.07 0.02 0.72 29 (44) EKLGETRVHQ 0.5 0.12 0.01 0.07 0.040.74 30 (45) ETRVHQALTE 0.42 0.16 0.03 0.05 0.03 0.69 31 (46) HQALTEVVIS0.43 0.21 0.04 0.1 0.05 0.83 32 (47) TEVVISSFDI 0.44 0.25 0.05 0.08 0.040.86 33 (48) ISSFDlKTNK 0.1 0.02 0.04 0.06 0.13 0.35 34 (49) DIKTNKPVIF0.57 0.22 0.04 0.18 0.28 1.29 35 (50) NKPVIFTKSN 0 0.01 0.02 0.07 0.240.34 36 (51) IFTKSNLANS 0 0 0.03 0.06 0.17 0.26 37 (52) SNLANSPELD 0.430.96 0.01 0.09 0.02 1.51 38 (53) NSPELDAKMY 0.18 0.12 0.01 0.05 0.050.41 39 (54) LDAKMYDISY 0.54 0.26 0.19 0.15 0.23 1.37 40 (55) MYDISYSTAA0.92 0.08 0.52 0.04 0.22 1.78 41 (56) SYSTAAAPTY 1.15 0.25 1.04 0.330.55 3.32 42 (57) AAAPTYFPPH 1.02 0.52 1.12 0.81 0.86 4.33 43 (58)TYFPPHYFVT 0.02 0.01 0.54 0.03 0.24 0.84 44 (59) PHYFVTNTSN 0.03 0.011.17 0.13 0.44 1.78 45 (60) VTNTSNGDEY 0.23 0.15 0.04 0.03 0.03 0.48 46(61) SNGDEYEFNL 0.33 0.25 0.08 0.1 0.11 0.87 47 (62) EYEFNLVDGA 0.340.25 0.07 0.1 0.2 0.96 48 (63) NLVDGAVATV 0.3 0.18 0.02 0.06 0.05 0.6149 (64) GAVATVADPA 0.45 0.54 0.01 0.07 0.02 1.09 50 (65) TVADPALLSI 0.480.29 0.01 0.07 0.03 0.88 51 (66) PALLSISVAT 0.65 0.33 0.02 0.1 0.01 1.1152 (67) SISVATRLAQ 0.61 0.23 0.14 0.53 0.53 2.04 53 (68) ATRLAQKDPA 0.870.34 0.05 0.29 0.22 1.77 54 (69) AQKDPAFASI 0.86 0.32 0.04 0.12 0.031.37 55 (70) PAFASIRSLN 0.81 0.15 0.05 0.51 0.59 2.11 56 (71) SIRSLNYKKM0.07 0.01 0.17 0.07 0.11 0.43 57 (72) LNYKKMLLLS 0.05 0.01 0.35 0.080.39 0.88 58 (73) KMLLLSLGTG 1.15 0.15 0.04 0.38 0.71 2.43 59 (74)LSLGTGTTSE 0.34 0.23 0.02 0.04 0.03 0.66 60 (75) TGTTSEFDKT 0.92 0.390.6 0.1 0.09 2.1 61 (76) SEFDKTYTAK 1.33 1.35 1.41 0.12 0.28 4.49 62(77) KTYTAKEAAT 1.36 0.94 1.11 0.76 0.4 4.57 63 (78) AKEAATWTAV 0.450.15 0.01 0.2 0.04 0.85 64 (79) ATWTAVHWML 0.1 0.02 0.01 0.08 0.06 0.2765 (80) AVHWMLVIQK 0.69 0.05 0.03 0.43 0.62 1.82 66 (81) MLVIQKMTDA 0.320.15 0.02 0.15 0.03 0.67 67 (82) QKMTDYYLST 0.26 0.125 0.03 0.21 0.050.675 68 (83) DAASSYMTDY 0.2 0.14 0.08 0.08 0.1 0.6 69 (84) SYMTDYYLST0.5 0.03 0.32 0.06 0.11 1.02 70 (85) DYYLSTAFQA 0.14 0 0.22 0.03 0.130.52 71 (86) STAFQALDSK 0.4 0.3 0.04 0.06 0.08 0.88 72 (87) QALDSKNNYL0.44 0.46 0.28 0.26 0.43 1.87 73 (88) SKNNYLRVQE 0.44 0.05 1.31 0.070.21 2.08 74 (89) YLRVQENALT 1.38 0.03 1.31 0.11 0.2 3.03 75 (90)QENALTGTTT 0.47 0.25 0 0.06 0 0.78 76 (91) LTGTTTEMDD 0.41 0.24 0 0.06 00.71 77 (92) TTEMDDASEA 0.38 0.3 0 0.05 0 0.73 78 (93) DDASEANMEL 0.440.24 0 0.06 0 0.74 79 (94) EANMELLVQV 0.42 0.27 0 0.04 0 0.73 80 (95)ELLVQVGENL 0.4 0.25 0 0.05 0 0.7 81 (96) QVGENLLKKP 0.44 0.14 0 0.07 00.65 82 (97) NLLKKPVSED 0.47 0.2 0 0.03 0 0.7 83 (98) KPVSEDNPET 0.270.21 0 0.03 0 0.51 84 (99) EDNPETYEEA 0.13 0.11 0 0.01 0 0.25 85 (100)ETYEEALKRF 1.26 1.2 1.36 0.53 0.71 5.06 86 (101) EALKRFAKLL 1.38 0.04 01.06 1.12 3.6 87 (102) RFAKLLSDRK 0.98 0.05 0 0.84 0.94 2.81 88 (103)LLSDRKKLRA 0.2 0.01 0 0.37 0.51 1.09 89 (104) RKKLRANKAS 0.28 0 0 0.310.64 1.23 Patient 41.84 20.565 18.1 14.17 16.55 Cumulative Totals

Example 4

Identification of Result Effective Substitutions

For each major and several minor allergenic epitopes of patatin, resulteffective substitutions were identified by synthesizing peptides thatwere altered by individually substituting an alanine residue at eachnon-alanine position in the epitope. Similarly, the reported nucleicacid sequence encoding corn patatin (U.S. Pat. No. 5,882,668; clone 5c9)was evaluated for IgE binding by producing peptides at correspondingpositions to the potato patatin protein.

For example, Epitope 41 was analyzed by alanine scanning and rationalsubstitution as follows.

Epitope 41 SEFDKTYTAK (SEQ ID NO:76) Alanine scan AEFDKTYTAK (SEQ IDNO:165) SAFDKTYTAK (SEQ ID NO:166) SEADKTYTAK (SEQ ID NO:167) SEFAKTYTAK(SEQ ID NO:168) SEFDATYTAK (SEQ ID NO:169) SEFDKAYTAK (SEQ ID NO:170)SEFDKTATAK (SEQ ID NO:171) SEFDKTYAAK (SEQ ID NO:172) SEFDKTYTAA (SEQ IDNO:173) Rational substitution AFFDKTYTAK (SEQ ID NO:283) SEFDKTFTAK (SEQID NO:176) Corn homolog CIFDSTYTAK (SEQ ID NO:284)

Selected epitopes were analyzed by alanine scanning and rationalsubstitution. Immunoassay with potato-allergic serum was used asdescribed above. Table 4 summarizes the results of these experiments toidentify result effective substitutions for patatin. Blank spaces in thetable indicate that binding of the peptide to patient IgE was notdetectable.

TABLE 4 Scanning of patatin for result effective substitutions Bindingof modified peptides by patient IgE as measured by OD Sequence SEQ ID NOHS03 HS06 HS01 HS02 DYFDVIGGTS 105 0.12 0.16 0.36 DYFDVIAGTS 106 0.140.17 0.4 VIGGTSTGGL 107 0.04 VIAGTSTGAL 108 AFYFEHGPQI 109 0.96 0.5 0.78PAYFEHGPQI 110 0.75 0.41 0.69 PFAFEHGPQI 111 PFYAEHGPQI 112 0.7 0.430.79 PFYFAHGPQI 113 0.93 1.07 0.59 1.44 PFYFEAGPQI 114 0.08 0.93 0.651.34 PFYFEHAPQI 115 0.75 0.54 1.11 PFYFEHGAQI 116 0.63 0.29 0.6PFYFEHGPAI 117 0.63 0.25 0.56 PFYFEHGPQA 118 0.27 0.16 0.33 TFYLENGPKI119 0.05 0.48 0.68 1.07 PFFFEHGPQI 120 AYLMQVLQEK 121 0.26 0.11 0.53KALMQVLQEK 122 KYAMQVLQEK 123 0.11 0.43 0.1 1.25 KYLAQVLQEK 124 0.220.48 0.11 1.34 KYLMAVLQEK 125 0.22 0.83 0.16 1.33 KYLMQALQEK 126 0.110.6 0.15 0.95 KYLMQVAQEK 127 0.53 0.15 0.81 KYLMQVLAEK 128 0.06 0.690.11 1.34 KYLMQVLQAK 129 0.74 0.79 0.05 0.58 KYLMQVLQEA 130 0.28 0.270.37 VFLHDKIKSL 131 0.06 0.26 0.41 AYSTAAAPTY 132 0.1 0.12 0.12SASTAAAPTY 133 SYATAAAPTY 134 0.16 0.13 0.37 SYSAAAAPTY 135 0.13 0.120.32 SYSTAAAATY 136 0.15 0.13 0.34 SYSTAAAPAY 137 0.15 0.14 0.29SYSTAAAPTA 138 0.55 0.54 1.13 CISTSAAPTY 139 0.4 SYSTAAAPAF 140 0.391.02 0.65 1.42 AFAAAAAPTY 141 0.07 SYSTAAAPTF 142 0.15 0.97 0.48 1.09STSAAPTYFP 143 0.21 0.23 0.39 STSAAPTFFP 144 0.23 STSAAPTAFP 145 0.08STAAAPTFFP 146 0.12 0.28 AAAATYFPPH 147 0.13 0.1 0.05 AAAPAYFPPH 1480.07 0.04 AAAPTAFPPH 149 AAAPTYAPPH 150 0.23 0.14 0.21 AAAPTYFAPH 1510.45 0.18 0.44 AAAPTYFPAH 152 0.15 0.07 0.18 AAAPTYFPPA 153 0.1 0.060.31 SAAPTYFPAH 154 0.77 0.73 0.96 AAAPAFFPPH 155 AAAPPFFPPH 156AAAPTFFPPH 157 SISVATRLAQ 158 0.26 0.26 AMSMLTKEVH 159 PAFASIRSLN 160PNFNAGSPTE 161 KMLLLSLGTG 162 NYLIISVGTG 163 0.49 1.08 0.64 1.48KMLLLSLGAG 164 0.13 AEFDKTYTAK 165 0.09 0.22 1.34 SAFDKTYTAK 166 0.660.71 0.06 1.42 SEADKTYTAK 167 0.99 SEFAKTYTAK 168 0.5 0.57 0.91SEFDATYTAK 169 0.17 SEFDKAYTAK 170 0.1 0.24 1.38 SEFDKTATAK 171 0.81SEFDKTYAAK 172 0.2 0.35 1.39 SEFDKTYTAA 173 0.1 1.18 KQAEKYTAEQ 174 0.080.24 SEFDAAFAAA 175 SEFDKTFTAK 176 0.09 0.16 0.07 1.45 AEKYTAEQCA 177ATYTAKEAAT 178 0.24 0.18 KAYTAKEAAT 179 0.28 0.33 KTATAKEAAT 180KTYAAKEAAT 181 0.1 0.32 0.73 KTYTAAEAAT 182 0.35 KTYTAKAAAT 183 0.4 0.590.82 KTYTAKEAAA 184 0.36 EKYTAEQCAK 185 AAFAAAEAAT 186 KTFTAKEAAT 187QALHCEKKYL 188 QALDSKAAYL 189 QALDSKNNFL 190 QALHCENNFL 191 CEKKYLRIQD192 1.01 0.16 SKNNFLRVQE 193 SENNYLRVQE 194 0.31 0.96 0.42 1 ALRVQENALT195 YARVQENALT 196 1.06 1.02 0.05 0.54 YLAVQENALT 197 0.37 1.04 0.111.06 YLRAQENALT 198 1.1 1 0.06 1.26 YLRVAENALT 199 1.03 0.92 0.08 1.26YLRVQANALT 200 1.05 0.92 0.06 1.24 YLRVQEAALT 201 0.93 0.92 0.07 1.11YLRVQENAAT 202 0.94 0.93 0.04 1.24 YLRVQENALA 203 1.05 0.96 0.43 1.16YLRIQDDTLT 204 1.07 0.85 0.39 1.12 YLTVAAAALT 205 1.05 0.86 0.28 1.33FLRVQENALT 206 NNYLRVQENA 207 0.23 0.88 0.5 1.17 KKYLRIQDDT 208 0.260.09 0.37 NNFLRVQENA 209 NAYLRVQENA 210 0.17 1.02 0.53 1.06 ATYEEAKLRF211 0.26 1.03 0.65 EAYEEALKRF 212 0.06 0.43 0.33 ETAEEALKRF 213 1.04ETYAEALKRF 214 0.62 1.02 1.15 ETYEAALKRF 215 1.06 0.38 0.89 ETYEEAAKRF216 0.08 0.1 0.9 ETYEEALARF 217 0.11 ETYEEALKAF 218 0.1 ETYEEALKRA 2190.1 GTNAQSLADF 220 ETYEAALAAF 221 0.07 0.78 0.33 0.77 ETFEEALKRF 222YEEALKTFAK 223 1.08 0.85 0.14 1.46 FEEALKRFAK 224 0.46 0.72 0.67AALKRFAKLL 225 0.15 0.17 EAAKRFAKLL 226 0.08 0.33 0.05 EALARFAKLL 2270.09 EALKAFAKLL 228 EALKRAAKLL 229 0.08 0.07 EALKRFAALL 230 EALKRFAKAL231 0.06 0.09 0.1 EALKRFAKLA 232 0.06 0.1 QSLADFAKQL 233 AALAAFAKLL 234LADFAKQLSD 235 DFAKQLSDER 236 0.17 AFAALLSDRK 237

Result effective substitutions were identified by a reduction in IgEbinding ability with respect to the non-substituted peptide sequence.Table 5 shows the identified result effective substitutions. Blankspaces in the table indicate that binding of the peptide to patient IgEwas not detectable. Many substitutions of alanine or phenylalanine forthe original tyrosine resulted in reduced or eliminated antibodybinding.

TABLE 5 Result effective substitutions of patatin Location (SEQ ID NO)Peptide (SEQ ID NO) HS03 HS06 HS01 HS02 Minor PFYFEHGPQI  (36) 1.08 0.170.65 0.06 Epitope ::A::::::: (111) 21 ::F::::::: (r) (120) :::::::::A(118) 0.27 0.16 0.33 Minor KYLMQVLQEK  (42) 1.08 0.79 0.12 0.04 Epitope:A:::::::: (122) 27 :::::::::A (130) 0.28 0.27 0.37 VFLHDKIKSL (c) (131)0.06 0.26 0.41 Major SYSTAAAPTY  (56) 1.04 0.55 1.15 0.25 EpitopeA::::::::: (132) 0.1 0.12 0.12 41 :A:::::::: (133) AFAA:::::: (r) (141)0.007 CI::S::::: (c) (139) 0.04 Overlap STAAAPTYFP (238) Epitope::S::::A:: (r) (145) 0.08 41/42 Major AAAPTYFPPH  (57) 1.12 0.86 1.020.52 Epitope ::::A::::: (148) 0.07 0.04 42 :::::A:::: (149) (57)::::AF:::: (r) (155) ::::PF:::: (r) (156) :::::F:::: (r) (157) MajorSEFDKTYTAK  (76) 0.12 0.28 1.33 1.35 Epitope ::::A::::: (169) 0.17 61KQAE:YTAEQ (c) (174) 0.08 0.24 ::::AAFA:A (r) (175) Major KTYTAKEAAT (77) 1.11 0.04 1.36 0.94 Epitope A::::::::: (178) 0.24 0.18 62::A::::::: (180) :::::A:::: (182) 0.35 AAFA:A:::: (r) (186) ::F:::::::(r) (187) EK:::EQC:K (c) (185) Minor QALDSKNNYL  (87) 0.28 0.43 0.440.46 Epitope :::HCEKK:: (c) (188) 72 ::::::AA:: (r) (189) ::::::::F: (r)(190) :::::E::F: (r) (240) Minor SKNNYLRVQE  (88) 1.31 0.21 0.44 0.05epitope ::F::::::: (r) (193) 73 Minor YLRVQENALT  (87) 1.31 0.2 1.380.03 epitope A::::::::: (195) 74 F::::::::: (r) (206) Overlap NNYLRVQENA(207) 0.23 0.88 0.5 1.17 epitope ::F::::::: (r) (209) 73/74 MajorETYEEALKRF (100) 1.36 0.71 1.26 1.2 epitope :::::::A:: (217) 0.11 85::::::::A: (218) 0.1 :::::::::A (219) 0.1 ::F::::::: (r) (222)G:NAQS:AD: (c) (220) Major EALKRFAKLL (101) 0 1.12 1.38 0.04 Epitope:::A:::::: (227) 0.09 86 ::::A::::: (228) :::::A:::: (229) 0.08 0.07:::::::A:: (230) ::::::::A: (231) 0.06 0.09 :::::::::A (232) 0.06SD:AD:::Q: (c) (241) A::AA::::: (r) (234) Epitope LKRFAKLLSD (239)overlap (NO 86/87 BINDING) Major RFAKLLSDRK (102) 0 0.94 0.98 0.05Epitope D:::Q:::ER (c) (236) 0.17 87 A::A:::::: (r) (237) (r) =rational; (c) = corn.

Example 5

Site Directed Mutagenesis

To introduce site specific mutations, the cloned DNA sequence of patatin(SEQ ID NO:1 encoding patatin protein SEQ ID NO:2; pMON 26820) wassubjected to PCR with primers SEQ ID NO:3 and SEQ ID NO:4 to incorporatepart of the α-factor signal sequence (Pichia expression manual,Invitrogen, Carlsbad, Calif.), and EcoRI and XhoI restriction sites tofacilitate cloning into the Pichia pastoris yeast secretion vector pPIC9(GenBank accession number Z46233; Invitrogen, Carlsbad, Calif.). TypicalPCR conditions are 25 cycles 94° C. denaturation for 1 minute, 45° C.annealing for one minute and 72° C. extension for 2 minutes; plus onecycle 72° C. extension for 10 minutes. A 50 μL reaction contains 30 pmolof each primer and 1 μg of template DNA; and 1×PCR buffer with MgCl2,200 μM dGTP, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 2.5 units of Pwo DNApolymerase. PCR reactions are performed in RoboCycler Gradient 96Temperature Cycler (Stratagene, La Jolla, Calif.).

The amplified fragment SEQ ID NO:5 was digested with restriction enzymesXhoI and EcoRI and cloned into the pBluescript vector (Stratagene, LaJolla, Calif.), digested with the same two restriction enzymes. Theresulting plasmid (pMON 26869) was used for oligonucleotide-directedmutagenesis using the Bio-Rad mutagenesis kit based on the method ofKunkel (Proc. Natl. Acad. Sci. U.S.A., 82: 477-492, 1985). Briefly,single-stranded pMON26869 was used as template for mutagenesis and wasprepared by superinfection of plasmid containing cells with M13K07(Gorman, et al., DNA Prot. Eng. Techniques, 2: 3-10, 1990). Themutagenic oligonucleotides are SEQ ID NOS:8-15 (reverse complement). DNApurified from transformed DH5αE. coli colonies was used for sequencedetermination. Sequencing was performed using the ABI PRISM sequencingkit (Perkin Elmer Biosystems, Foster City, Calif.). The resultingplasmid containing the mutation in the patatin gene was digested withrestriction enzymes XhoI and EcoRI.

The patatin nucleic acid fragment was then ligated into the pPIC9 vector(Invitrogen, Carlsbad, Calif.), digested with the same two restrictionenzymes to afford plasmid pMON37401. Pichia pastoris KM71 cells wereelectroporated with pMON37401 containing the appropriate mutation. Theresulting transformed cells were used to produce protein in Pichiapastoris using the procedure supplied by the manufacturer (Invitrogen,Carlsbad, Calif.). The encoded protein contains an alpha factor signalcleavage site. Plasmid pMON37401 encodes SEQ ID NO:6 which is cleaved toafford SEQ ID NO:7, having four amino acids added at the N-terminus ofamino acids 24-386 of SEQ ID NO:2. Position four of SEQ ID NO:7therefore corresponds to position 23 of SEQ ID NO:2.

The concentration of patatin in the culture was determined using apatatin ELISA assay and the enzyme activity was measured using themethod of Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990). Thevariants containing multiple mutations were further purified using MonoQ and hydrophobic interaction chromatography (HIC). Each culture waspurified by first sizing on Amicon YM10 membranes (Millipore, Bedford,Mass.) to a >10 kDa fraction, followed by chromatography on the Mono QHR 10/10 column (Pharmacia, Piscataway, N.J.). For chromatography on theMono Q column, the samples were loaded on the column in 25 mM Tris pH7.5 and eluted with a gradient of 1.0 M KCl in 25 mM Tris pH 7.5.Fractions containing patatin protein were determined using SDS-PAGE. Forchromatography on the HIC column, the appropriate fractions were pooledand dialyzed into 1 M ammonium sulfate in 25 mM Tris pH 7.5. Thedialyzed sample was then loaded on 16/10 phenyl Sepharose column(Pharmacia, Piscataway, N.J.) and eluted with a gradient of 25 mM TrispH7.5.

The protein concentration was determined using the Bradford method,using BSA as a standard. SDS-PAGE analysis showed that these proteinswere essentially pure. The esterase activity of the newly formedvariants are shown in Table 6. The activity was determined usingp-nitrophenyl caprate substrate as described by Hofgen and Willmitzer(Plant Science, 66: 221-230, 1990).

TABLE 6 Esterase activity of patatin mutants Activity Variant (mOD ·min⁻¹μg⁻¹) Wild type 93.2 Y106F 51.1 Y129F 74.7 Y185F 85.6 Y193F 82.2Y185F/Y193F 99.4 Y270F 163.4 Y316F 94.88 Y362F 130.7Y106F/Y129F/Y185F/Y193F/Y270F/Y316F/Y362F 57.1Y185F/Y193F/Y270F/Y316F/Y362F 161.5

Patatin proteins having a phenylalanine substitution at each of theamino acid positions 106, 129, 185, 193, 270, 316 and 362 (numberscorrespond to positions in SEQ ID NO:2) of expressed SEQ ID NO:7 exhibitfull enzyme activity. Proteins having multiple substitutions alsodisplayed full enzyme activity.

In addition to nucleotide sequences encoding conservative amino acidchanges within the fundamental polypeptide sequence, biologicallyfunctional equivalent nucleotide sequences include nucleotide sequencescontaining other base substitutions, additions, or deletions. Theseinclude nucleic acids containing the same inherent genetic informationas that contained in the cDNA which encode peptides, polypeptides, orproteins conferring pathogen resistance the same as or similar to thatof pathogen upon host cells and plants. Such nucleotide sequences can bereferred to as “genetically equivalent modified forms” of the cDNA, andcan be identified by the methods described herein.

Mutations made in the cDNA, plasmid DNA, genomic DNA, synthetic DNA, orother nucleic acid encoding the deallergenized gene preferably preservethe reading frame of the coding sequence. Furthermore, these mutationspreferably do not create complementary regions that could hybridize toproduce secondary mRNA structures, such as loops or hairpins, that wouldadversely affect mRNA translation.

Although mutation sites can be predetermined, it is not necessary thatthe nature of the mutations per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis can be conducted at the target codon.

Alternatively, mutations can be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native cDNAsequence. Following ligation, the resulting reconstructed nucleotidesequence encodes a derivative form having the desired amino acidinsertion, substitution, or deletion.

Example 6

Construction of Permutein Sequences

Nucleic acid sequences encoding permutein proteins having rearrangedN-terminus/C-terminus protein sequences can be made by following thegeneral method described by Mullins et al. (J. Am. Chem. Soc. 116:5529-5533, 1994). The steps are shown in FIG. 3. The Figure and thefollowing Examples involve the design and use of a linker regionseparating the original C-terminus and N-terminus, but the use of alinker is not a critical or required element of permutein design.

Two sets of oligonucleotide primers are used in the construction of anucleic acid sequence encoding a permutein protein. In the first step,oligonucleotide primers “new N-termini” and “linker start” are used in aPCR reaction to create amplified nucleic acid molecule “new N-terminifragment” that contains the nucleic acid sequence encoding the newN-terminal portion of the permutein protein, followed by the polypeptidelinker that connects the C-terminal and N-terminal ends of the originalprotein. In the second step, oligonucleotide primers “new C-termini” and“linker end” are used in a PCR reaction to create amplified nucleic acidmolecule “new C-termini fragment” that contains the nucleic acidsequence encoding the same linker as used above, followed by the newC-termini portion of the permutein protein. The “new N-termini” and “newC-termini” oligonucleotide primers are designed to include appropriaterestriction enzyme recognition sites which assist in the cloning of thenucleic acid sequence encoding the permutein protein into plasmids.

Any suitable PCR conditions and polymerase can be used. It is desirableto use a thermostable DNA polymerase with high fidelity to reduce oreliminate the introduction of sequence errors. Typical PCR conditionsare 25 cycles 94° C. denaturation for 1 minute, 45° C. annealing for oneminute and 72° C. extension for 2 minutes; plus one cycle 72° C.extension for 10 minutes. A 50 μL reaction contains 30 pmol of eachprimer and 1 μg of template DNA; and 1×PCR buffer with MgCl₂, 200 μMdGTP, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 2.5 units of Pwo DNApolymerase. PCR reactions are performed in RoboCycler Gradient 96Temperature Cycler (Stratagene, La Jolla, Calif.).

The amplified “new N-termini fragment” and “new C-termini fragment” areannealed to form a template in a third PCR reaction to amplify thefull-length nucleic acid sequence encoding the permutein protein. TheDNA fragments “new N-termini fragment” and “new C-termini fragment” areresolved on a 1% TAE gel, stained with ethidium bromide, and isolatedusing the QIAquick Gel Extraction Kit (Qiagen, Valencia, Calif.). Thesefragments are combined in equimolar quantities with oligonucleotideprimers “new N-termini” and “new C-termini” in the third PCR reaction.The conditions for the PCR are the same as used previously. PCR reactionproducts can be purified using the QIAquick PCR purification kit(Qiagen, Valencia, Calif.).

Alternatively, a linker sequence can be designed containing arestriction site, allowing direct ligation of the two amplified PCRproducts.

Example 7

Construction of Plasmid pMON 37402

The patatin protein contains a trypsin protease sensitive site at thearginine amino acid at position 246, as determined by electrophoresis ofa trypsin digest reaction. In order to determine if the exposed proteasesite is an antigenic epitope, a permutein was constructed usingpositions 246-247 as a breakpoint.

The nucleic acid sequence encoding the permutein protein in plasmid pMON37402 was created using the method illustrated in FIG. 3 and describedin Example 6. Nucleic acid molecule “new N-termini fragment” was createdand amplified from the sequence encoding patatin in plasmid pMON26820using oligonucleotide primers 27 (SEQ ID NO:242) and 48 (SEQ ID NO:243).Nucleic acid molecule “new C-termini fragment” was created and amplifiedfrom the sequence encoding patatin in plasmid pMON26820 usingoligonucleotide primers 47 (SEQ ID NO:244) and 36 (SEQ ID NO:245). Thefull-length nucleic acid molecule encoding the permutein protein wascreated and amplified from annealed fragments “new N-termini fragment”and “new C-termini fragment” using oligonucleotide primers 27 (SEQ IDNO:242) and 36 (SEQ ID NO:245).

The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on ampicillin-containing plates. Plasmid DNA was isolated andsequenced to confirm the presence of the correct insert. The resultingplasmid was designated pMON 37402 (containing SEQ ID NO:246, encodingprotein sequence SEQ ID NO:247).

Example 8

Construction of Plasmid pMON 37405

Amino acids 201-202, near tyrosine 193, were chosen as a breakpoint forthe construction of a permutein protein.

The nucleic acid sequence encoding the permutein protein in plasmid pMON37405 was created using the method illustrated in FIG. 3 and describedin Example 6. Nucleic acid molecule “New N-termini fragment” was createdand amplified from the sequence encoding patatin in plasmid pMON26820using oligonucleotide primers 48 (SEQ ID NO:243) and 58 (SEQ ID NO:249).Nucleic acid molecule “New C-termini fragment” was created and amplifiedfrom the sequence encoding patatin in plasmid pMON26820 usingoligonucleotide primers 47 (SEQ ID NO:244) and 59 (SEQ ID NO:249). Thefull-length nucleic acid molecule encoding the permutein protein wascreated and amplified from annealed fragments “New N-termini fragment”and “New C-termini fragment” using oligonucleotide primers 58 (SEQ IDNO:248) and 59 (SEQ ID NO:249).

The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on ampicillin-containing plates. Plasmid DNA was isolated andsequenced to confirm the presence of the correct insert. The resultingplasmid was designated pMON 37405 (containing SEQ ID NO:250, encodingprotein sequence SEQ ID NO:251).

Example 9

Construction of Plasmid pMON 37406

Amino acids 183-184, adjacent to tyrosine 185, were chosen as abreakpoint for the construction of a permutein protein.

The nucleic acid sequence encoding the permutein protein in plasmid pMON37406 was created using the method illustrated in FIG. 3 and describedin Example 6. Nucleic acid molecule “New N-termini fragment” was createdand amplified from the sequence encoding patatin in plasmid pMON26820using oligonucleotide primers 48 (SEQ ID NO:243) and 60 (SEQ ID NO:252).Nucleic acid molecule “New C-termini fragment” was created and amplifiedfrom the sequence encoding patatin in plasmid pMON26820 usingoligonucleotide primers 47 (SEQ ID NO:244) and 61 (SEQ ID NO:253). Thefull-length nucleic acid molecule encoding the permutein protein wascreated and amplified from annealed fragments “New N-termini fragment”and “New C-termini fragment” using oligonucleotide primers 60 (SEQ IDNO:252) and 61 (SEQ ID NO:253).

The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on ampicillin-containing plates. Plasmid DNA was isolated andsequenced to confirm the presence of the correct insert. The resultingplasmid was designated pMON37406 (containing SEQ ID NO:254, encodingprotein sequence SEQ ID NO:255).

Example 10

Construction of Plasmid pMON 37407

Amino acids 268-269, adjacent to tyrosine 270, were chosen as abreakpoint for the construction of a permutein protein.

The nucleic acid sequence encoding the permutein protein in plasmid pMON37407 was created using the method illustrated in FIG. 3 and describedin Example 6. Nucleic acid molecule “New N-termini fragment” was createdand amplified from the sequence encoding patatin in plasmid pMON26820using oligonucleotide primers 48 (SEQ ID NO:243) and 62 (SEQ ID NO:256).Nucleic acid molecule “New C-termini fragment” was created and amplifiedfrom the sequence encoding patatin in plasmid pMON26820 usingoligonucleotide primers 47 (SEQ ID NO:244) and 63 (SEQ ID NO:257). Thefull-length nucleic acid molecule encoding the permutein protein wascreated and amplified from annealed fragments “New N-termini fragment”and “New C-termini fragment” using oligonucleotide primers 62 (SEQ IDNO:256) and 63 (SEQ ID NO:257).

The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on ampicillin-containing plates. Plasmid DNA was isolated andsequenced to confirm the presence of the correct insert. The resultingplasmid was designated pMON37407 (containing SEQ ID NO:258, encodingprotein sequence SEQ ID NO:259).

Example 11

Construction of Plasmid pMON 37408

Amino acids 321-322, near tyrosine 216, were chosen as a breakpoint forthe construction of a permutein protein.

The nucleic acid sequence encoding the permutein protein in plasmid pMON37408 was created using the method illustrated in FIG. 3 and describedin Example 6. Nucleic acid molecule “New N-termini fragment” was createdand amplified from the sequence encoding patatin in plasmid pMON26820using oligonucleotide primers 48 (SEQ ID NO:243) and 64 (SEQ ID NO:260).Nucleic acid molecule “New C-termini fragment” was created and amplifiedfrom the sequence encoding patatin in plasmid pMON26820 usingoligonucleotide primers 47 (SEQ ID NO:244) and 65 (SEQ ID NO:261). Thefull-length nucleic acid molecule encoding the permutein protein wascreated and amplified from annealed fragments “New N-termini fragment”and “New C-termini fragment” using oligonucleotide primers 64 (SEQ IDNO:260) and 65 (SEQ ID NO:261).

The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on ampicillin-containing plates. Plasmid DNA was isolated andsequenced to confirm the presence of the correct insert. The resultingplasmid was designated pMON37408 (containing SEQ ID NO:262, encodingprotein sequence SEQ ID NO:263).

Example 12

Production of Permutein Proteins in Pichia pastoris

Plasmids pMON37402, pMON37405, pMON37406, pMON37407, and pMON37408 wereindividually used to electroporate KM71 cells from Pichia pastorisaccording to the procedure supplied by the manufacturer (Invitrogen,Carlsbad, Calif.). The resulting transformed cells were used to produceprotein in Pichia pastoris following the procedure supplied by themanufacturer (Invitrogen, Carlsbad, Calif.).

The concentration of patatin in the culture was determined using apatatin ELISA assay and the enzyme activity was measured using themethod of Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990). Thevariants containing multiple mutations were further purified using MonoQ and hydrophobic interaction chromatography (HIC). Each culture waspurified by first sizing on YM10 membranes (Amicon, Mass.) to a [>10kDa] fraction, followed by chromatography on the Mono Q HR 10/10 column(Pharmacia, N.J.). For chromatography on the Mono Q column, the sampleswere loaded on the column in 25 mM Tris pH 7.5 and eluted with agradient of 1.0 M KCl in 25 mM Tris pH 7.5. Fractions containing patatinprotein were determined using SDS-PAGE. For chromatography on the HICcolumn, the appropriate fractions were pooled and dialyzed into 1 Mammonium sulfate in 25 mM Tris pH 7.5. The dialyzed sample was thenloaded on 16/10 phenyl Sepharose column (Pharmacia, N.J.) and elutedwith a gradient of 25 mM Tris pH7.5.

The protein concentration was determined using the Bradford method,using BSA as a standard. SDS-PAGE analysis showed that these proteinswere essentially pure. The esterase activity of the variants are shownin Table 7.

TABLE 7 Activity of permuteins pMON Breakpoint Activity (ΔOD min⁻¹μg⁻¹)Native enzyme  83.21 pMON37402 246/247 66.7 pMON37405 201/202 Noexpression pMON37406 183/184 No expression pMON37407 268/269 12.1pMON37408 321/322 No expression

The activity was determined using p-nitrophenyl caprate substrate asdescribed by Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990).

Example 13

Insect Bioefficacy Assays

Assays for activity against larvae of SCRW are carried out by overlayingthe test sample on an agar diet similar to that described by Marrone (J.Econ. Entom. 78: 290-293, 1985). Test samples were prepared in 25 mMTris, pH 7.5 buffer. Neonate larvae are allowed to feed on the treateddiet at 26° C., and mortality and growth stunting were evaluated after 5or 6 days. The results of this assay are shown in Table 8.

TABLE 8 Insect bioefficacy assay Protein (200 ppm) Mean Survival Weight% Weight Reduction Tris buffer (control) 1.26 ± 0.3  — Wild Type 0.21 ±0.02 83 pMON37402 0.21 ± 0.03 83 pMON37407 0.32 ± 0.04 75

These data demonstrate that the growth of the SCRW larvae is similarlyreduced upon ingestion of the proteins encoded by pMON37402 andpMON37407 as compared to the wild type patatin protein.

Example 14

Permutein Sequences Improved for Monocot Expression

Modification of coding sequences has been demonstrated above to improveexpression of insecticidal proteins. A modified coding sequence was thusdesigned to improve expression in plants, especially corn (SEQ IDNO:264).

Example 15

Construction of pMON40701 for Monocot Expression

Plasmid pMON19767 was digested with restriction endonucleases NcoI andEcoRI and the 1100 bp gene fragment was purified using the QIAquick PCRpurification kit (Qiagen, Valencia, Calif.). Plasmid pMON33719 wasdigested with restriction endonucleases NcoI and EcoRI, and gelpurified, resulting in an approximately 3900 base pair vector fragment.The two purified restriction fragments were combined and ligated usingT4 DNA ligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on ampicillin-containing plates. Plasmid DNA was isolated andsequenced to confirm the presence of the correct insert. The resultingplasmid was designated pMON40700. Plasmid pMON40700 was digested withrestriction endonuclease NotI and the resulting 2200 bp DNA fragment waspurified using the QIAquick PCR purification kit (Qiagen, Valencia,Calif.). Plasmid pMON30460 was digested with restriction endonucleaseNotI, and gel purified, resulting in an approximately 4200 base pairvector fragment. The two purified restriction fragments were combinedand ligated using T4 DNA ligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on kanamycin-containing plates. The resulting plasmid wasdesignated pMON40701 (containing SEQ ID NO:264, encoding proteinsequence SEQ ID NO:265).

Example 16

Construction of pMON40703 for Monocot Expression

The nucleic acid sequence encoding the permutein protein in plasmidpMON40703 was created using the method illustrated in FIG. 3 anddescribed in Example 6. Nucleic acid molecule “New N-termini fragment”was created and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn1 (SEQ ID NO:266) and Syn2(SEQ ID NO:267). Nucleic acid molecule “New C-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:268) and Syn4(SEQ ID NO:269). The full-length nucleic acid molecule encoding thepermutein protein was created and amplified from annealed fragments “NewN-termini fragment” and “New C-termini fragment” using oligonucleotideprimers Syn1 (SEQ ID NO:266) and Syn4 (SEQ ID NO:269).

The resulting amplified nucleic acid molecule was digested withrestriction endonucleases NcoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). PlasmidpMON33719 was digested with restriction endonucleases NcoI and EcoRI,and gel purified, resulting in an approximately 3900 base pair vectorfragment. The purified restriction fragments were combined and ligatedusing T4 DNA ligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on ampicillin-containing plates. Plasmid DNA was isolated andsequenced to confirm the presence of the correct insert. The resultingplasmid was designated pMON40702. Plasmid pMON40702 was digested withNotI, and the resulting 2200 bp DNA fragment was purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). PlasmidpMON30460 was digested with restriction endonuclease NotI, and gelpurified, resulting in an approximately 4200 base pair vector fragment.The purified restriction fragments were combined and ligated using T4DNA ligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on kanamycin-containing plates. The resulting plasmid wasdesignated pMON40703 (containing SEQ ID NO:270, encoding proteinsequence SEQ ID NO:271). Plasmid pMON40703 encodes a permutein proteinwith a “breakpoint” at positions 246/247 of the wild type patatinprotein sequence (SEQ ID NO:2). The first 23 amino acids of SEQ ID NO:2are a signal peptide sequence which is cleaved in the mature protein.

Example 17

Construction of pMON40705 for Monocot Expression

The nucleic acid sequence encoding the permutein protein in plasmidpMON40705 was created using the method illustrated in FIG. 3 anddescribed in Example 6. Nucleic acid molecule “New N-termini fragment”was created and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn10 (SEQ ID NO:272) and Syn2(SEQ ID NO:267). Nucleic acid molecule “New C-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:268) and Syn11(SEQ ID NO:273). The full-length nucleic acid molecule encoding thepermutein protein was created and amplified from annealed fragments “NewN-termini fragment” and “New C-termini fragment” using oligonucleotideprimers Syn10 (SEQ ID NO:272) and Syn11 (SEQ ID NO:273).

The resulting amplified nucleic acid molecule was digested withrestriction endonucleases NcoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). PlasmidpMON33719 was digested with restriction endonucleases NcoI and EcoRI,and gel purified, resulting in an approximately 3900 base pair vectorfragment. The purified restriction fragments were combined and ligatedusing T4 DNA ligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on ampicillin-containing plates. Plasmid DNA was isolated andsequenced to confirm the presence of the correct insert. The resultingplasmid was designated pMON40704. Plasmid pMON40704 was digested withrestriction endonuclease NotI, and the resulting 2200 bp DNA fragmentwas purified using the QIAquick PCR purification kit (Qiagen, Valencia,Calif.). Plasmid pMON30460 was digested with restriction endonucleaseNotI, and gel purified, resulting in an approximately 4200 base pairvector fragment. The purified restriction fragments were combined andligated using T4 DNA ligase.

The ligation reaction mixture was used to transform E. coli strain DH5αcells (Life Technologies, Gaithersburg, Md.). Transformant bacteria wereselected on plates containing kanamycin. The resulting plasmid wasdesignated pMON40705 (containing SEQ ID NO:274, encoding proteinsequence SEQ ID NO:275). Plasmid pMON40705 encodes a permutein proteinwith a “breakpoint” at positions 268/269 of the wild type patatinprotein sequence (SEQ ID NO:2). The first 23 amino acids of SEQ ID NO:2are a signal peptide sequence which is cleaved in the mature protein.

Example 18

Transient Expression of Protein in Corn Leaf Protoplasts

Plasmids pMON40701, pMON40703, and pMON40705 (all containing the nativesignal sequence for vacuolar targeting) were separately electroporatedinto corn leaf protoplasts as described by Sheen (Plant Cell 3: 225-245,1991). Protein was extracted with glass beads and the supernatant wasassayed for protein expression using ELISA for patatin and NPTII.Expression of protein by the transformed corn protoplasts was confirmedby Western blot analysis. Expression results are shown in Table 9.

TABLE 9 ELISA data Normalized Expression Patatin ELISA NPTII ELISA(Patatin ELISA/ Sample (μg/mL) (μg/mL) NPTII ELISA) pMON40701 1.1 0.61.8 pMON40703 2.1 0.3 7.0 pMON40705 1.3 0.6 2.2

The results indicate that the permutein encoded by plasmid pMON40703surprisingly shows approximately 4-fold higher expression compared tothe wild type enzyme.

Example 19

Deglycosylation of Protein Sequences

This example provides evidence that glycosylation of can contribute tothe allergenicity of a protein. Accordingly, rational substitution ofamino acid residues likely to be the targets of glycosylation within asubject allergen protein may reduce or eliminate the allergenicproperties of the protein without adversely affecting the enzymatic,insecticidal, antifungal or other functional properties of the protein.

Glycosylation commonly occurs as either N-linked or O-linked forms.N-linked glycosylation usually occurs at the motif Asn-Xaa-Ser/Thr,where Xaa is any amino acid except Pro (Kasturi, L. et al., Biochem J.323: 415-519, 1997; Melquist, J. L. et al., Biochemistry 37: 6833-6837,1998). O-linked glycosylation occurs between the hydroxyl group ofserine or threonine and an amino sugar.

Site directed mutagenesis of selected asparagine, serine, or threoninemay be used to reduce or eliminate the glycosylation of patatinproteins. A search of SEQ ID NO:2 for the Asn-Xaa-Ser/Thr motif revealsone occurrence at amino acid positions 202-204. Mutagenization of thenucleic acid sequence encoding this region results in a reducedallergenicity of the encoded protein.

In order to test this approach to reducing allergenicity of patatinproteins, two sets of experiments were performed: a) production ofpatatin proteins in Escherichia coli, which do not glycosylate proteins;and b) production of patatin proteins with an N202Q site directedmutation.

Antibodies obtained from patients HS-07 and G15-MON (not potatoallergic) did not show specific binding to wild type patatin, patatinproduced in E. coli, or the N202Q variant. Antibodies obtained frompatient HS-01 (potato allergic) bound to wild type patatin, but not topatatin produced in E. coli or the N202Q variant. Antibodies obtainedfrom patient HS-02 (potato allergic) bound strongly to wild typepatatin, but extremely weakly to patatin produced in E. coli, andbinding to the N202Q variant resembled vector controls. Antibodiesobtained from patient HS-03 (potato allergic) bound to wild typepatatin, but not to patatin produced in E. coli or the N202Q variant.Antibodies obtained from patient HS-05 (potato allergic) bound to wildtype patatin, but very weakly to patatin produced in E. coli and theN202Q variant. Antibodies obtained from patient HS-06 (potato allergic)strongly bound wild type patatin, the N202Q variant, and to patatinproduced in E. coli. These results strongly suggest that glycosylationis at least partially responsible for the antigenic properties ofpatatin proteins, and that site directed mutagenesis may be used toreduce or eliminate specific antibody binding. Mutagenesis at position202 of SEQ ID NO:2 may be useful for reducing or eliminating specificantibody binding.

The deglycosylation approach was also tested using a patatin homolog,Pat17. As demonstrated above, patatin epitopes exhibiting IgE bindingwere identified, and each contained a Tyr residue. Substitution of theseTyr residues within each epitope led to loss of IgE binding.Site-directed mutagenesis was used to produce variants with individualand multiple Tyr substitutions in the protein, which was expressed inPichia pastoris and assessed for enzyme activity. All the variants werefound to have enzymatic activity no less than the wild type protein. Asingle variant with all 5 tyrosine residues substituted withphenylalinine was found to have insecticidal activity no less than theunsubstituted protein and was expressed in E. coli to produce thenon-glycosylated version. The E. coli 5-“Tyr to Phe” variant wasassessed for IgE binding. An isozyme of patatin, designated Pat17, wasalso expressed in corn to produce a plant glycoprotein and in E. coli toproduce a nonglycosylated protein. Sera of seven patients (fiveexhibiting potato allergy and one exhibiting other allergies but noallergy to potatoes) were were used to assay Pat17 or Pat17 variantbinding by immunoblot assay. Four of the five sera from patientsexhibiting potato allergy showed either very weak or no binding to wildtype patatin expressed in E. coli but did bind to the 5-Tyr variant.Serum from one patient exhibiting potato allergy showed strong bindingto recombinant wild type patatin protein expressed in E. coli but weakbinding to the 5-Tyr variant. Sera from all five patients exhibitingpotato allergy bound strongly to patatin expressed in corn and nativepatatin present in potatoes. Serum from a control patient allergic toeggs, milk, peanuts and seafood, but exhibiting no allergy to potatoesshowed no binding to patatin expressed in E. coli but did bind topatatin expressed in corn. Immunoblot results suggested that the sugarmoiety in patatin is a non-specific IgE binding epitope and thepolypeptide portion of patatin also contains immunogenic IgE epitopes.

Patients who suffer from potato allergy were identified at Johns HopkinsClinic (Baltimore, Md.) and were evaluated for potato allergy usingclinical criteria outlined in Table 2.

Serum was obtained from patients with convincing clinical history ofpotato allergy. The convincing history was defined as being one or moreof the following: a) positive potato allergic reaction as evaluated bydouble-blind placebo-control food challenge b) anaphylaix and/orhospitalization due to the consumption of potatoes or c) dramatic skintest results.

Peptide Synthesis

Peptides were synthesized on cellulose membranes using the SPOTS system(Genosys Biotechnologies, TX). Membranes were stored at −20° C. untiluse.

Site Directed Mutagenesis

Site specific mutations were introduced into patatin by firstincorporating part of the a-factor signal sequence (Pichia expressionmanual, Invitrogen, Carlsbad, Calif.) to the patatin gene using PCR.Primers used for the PCR wereGGAGCTCGAGAAAAGAGAGGCTGAAGCTCAGTTGGGAGAAATGGTGACTGTTCT (SEQ ID NO: 3)(XhoI site in italics) and GGTCTAGAG GAATTCTCATTAATAAGAAG (SEQ ID NO: 4)(EcoRI site in italics). The primers contained restriction sites tofacilitate cloning into Pichia pastoris yeast secretion vector pPIC9(GenBank accession number Z46233; Invitrogen, Carlsbad, Calif.). TypicalPCR conditions are 25 cycles 94° C. denaturation for 1 minute, 45° C.annealing for one minute and 72° C. extension for 2 minutes; plus onecycle 72° C. extension for 10 minutes. A 50 mL reaction contained 30pmol of each primer and 1 mg of template DNA; and 1×PCR buffer withMgCl₂, 200 mM dGTP, 200 mM dATP, 200 mM dTTP, 200 mM dCTP, 2.5 units ofPwo DNA polymerase. PCR reactions are performed in RoboCycler Gradient96 Temperature Cycler (Stratagene, La Jolla, Calif.).

The amplified patatin gene was digested with restriction enzymes XhoIand EcoRI and cloned into the pBluescript vector (Stratagene, La Jolla,Calif.), digested with the same two restriction enzymes. The templateplasmid DNA used for the PCR was pMON26820. The resulting plasmid (pMON26869) was used for oligonucleotide-directed mutagenesis using theBio-Rad mutagenesis kit based on the method of Kunkel et al., Proc NatlAcad Sci USA 82, 477-92 (1985). Briefly, single-stranded pMON26869 wasused as template for mutagenesis and was prepared by superinfection ofplasmid containing cells with M13K07 (Gorman et al., DNA and ProteinEngineering techniques 2, 3-10 (1990)). DNA purified from transformedDH5a E. coli colonies was used for sequence determination. Sequencingwas performed using the ABI PRISM sequencing kit (Perkin ElmerBiosystems, Foster City, Calif.).

Protein Expression in Pichia pastoris

Plasmids containing the mutations in the patatin gene were digested withrestriction enzymes XhoI and EcoRI. The patatin nucleic acid fragmentwas then ligated into the pPIC9 vector (Invitrogen, Carlsbad, Calif.),digested with the same two restriction enzymes to afford plasmidpMON37401. Pichia pastoris KM71 cells were electroporated with pMON37401containing the appropriate mutation. The resulting transformed cellswere used to produce protein in Pichia pastoris using the proceduresupplied by the manufacturer (Invitrogen, Carlsbad, Calif.). Theproteins were purified in the same way as the proteins expressed in E.coli (see below).

Western Blotting of Proteins

Protein samples were electrophoresed by SDS-PAGE and electroblotted ontoPVDF membrane (Millipore, Bedford Mass.). Protein blots were processedby standard Western blotting (immunoblotting) techniques and wereincubated in potato allergic serum diluted 1:5 in PBS buffer for 1 hour.After washing the blots 3 times with PBS, the blots were incubated inbiotinylated anti-IgE (Johns Hopkins Hospital, Baltimore Md.) for 1hour, followed by a 30 minute incubation in HRP-linked avidin (Promega,New York, N.Y.). IgE-reactive protein bands were visualized by using theECL system (Amersham Pharmacia Biotech, N.J.). As a control, one blotwas incubated in anti-IgE only. His-tagged glyphosate oxidase and potatoextracts was prepared and provided for this study by RegulatorySciences, Monsanto Company. The peptides were evaluated using the sameincubation procedures as described above.

Expression and Purification of Patatin in Corn

An isozyme of patatin, Pat17, was generated for expression in corn usinga modified plant optimized gene sequence as described by Brown et al(U.S. Pat. No. 5,689,052). All the constructs contained the native 23amino acid signal peptide for vacuolar targeting. Corn was transformedby microprojectile bombardment (Morrish et al., in Transgenic plants.Fundamentals and Applications (ed. Hiatt, A.) 133-171 (Marcel Dekker,New York, 1993); Songstad et al., In Vitro Cell Dev Biol—Plant 32,179-183 (1996)). Protein from the transformed corn plants was purifiedby first grinding the leaves in liquid nitrogen and extracting theprotein using 25 mM Tris/HCl. The plant extract was further dialyzedagainst 25 mM Tris/HCl pH 7.5. The plant extract was then loaded ontoMono Q HR 10/10 anion-exchange column (Amersham Pharmacia, N.J.)equilibrated with 25 mM Tris/HCl pH 7.5 (buffer A). The protein waseluted with 25 mM Tris/HCl pH 7.5, 1 M KCl (buffer B) using a lineargradient of 0-100% buffer B using an HPLC system (Shimadzu). Fractionscontaining protein were assayed for esterase activity and dialyzedagainst 25 mM Tris/HCl pH 7.5, 1 M Ammonium Sulfate (buffer C). Theprotein was purified to homogeneity by loading onto a phenyl-Sepharose16/10 column (Amersham Pharmacia, N.J.) equilibrated with buffer C.Esterase active fractions were pooled and dialyzed against 25 mMTris/HCl pH 7.5.

Expression and Purification of Patatin in E. coli

Pat17 was expressed in E. coli using the pET expression system (Novagen,Wis.). The coding region of the mature Pat17 gene (without its signalpeptide) was amplified by PCR using the primers5′-GGGCCATGGCGCAGTTGGGAGAAATGGTG-3′ (SEQ ID NO: 294) (NcoI site initalics) and 5′-AACAAAGCTTCTTATTGAGGTGCGGCCGCTTGCATGC-3′ (SEQ ID NO:295) (NotI site in italics) using standard PCR reaction conditions asdescribed in the Gene Amp kit (Perkin-Elmer Cetus, Conn.) and anannealing temperature of 40° C. The template was plasmid pMON26820. Theresulting DNA was digested with NcoI and NotI and cloned into a modifiedpET24d plasmid, designed to add an N-terminal hexa-histidine tag to theprotein. The correct sequence of the PCR product was verified bysequencing, and the plasmid was transformed into E.coli BL21 (DE3), andtransformants selected on LB containing 25 mg/mL kanamycin. Theexpression strain was grown in LB containing 25 mg/mL kanamycin andinduced for 8 hrs at 28° C. with 1 mM IPTG. Cells were harvested andwashed in 50 mM Tris/HCl pH 8.5, 150 mM NaCI, and lysed by French Pressat 20,000 psi. His-tagged protein was recovered in the soluble fractionof lysed cells and subsequently purified using Ni—NTA resin as describedin the QIAexpressionist manual (Qiagen Calif.). The partially purifiedprotein was then dialyzed against 25 mM Tris/HCl pH 7.5 (buffer A) andloaded onto Mono Q HR 10/10 anion-exchange column (Amershani Pharmacia,N.J.) equilibrated with buffer A. The protein was eluted with 25 mMTris/HCl pH 7.5, 1 M KCl (buffer B) using a linear gradient of 0-100%buffer B run over 30 min at a flow rate of 4 mL/min using an HPLC system(Shimadzu). Fractions containing protein were assayed for esteraseactivity. Esterase active fractions were pooled, concentrated anddialyzed against 25 mM Tris/HCl pH 7.5 and stored at 4° C.

Enzyme Activity Assays

Enzyme activity was measured as described previously using p-nitrophenylcaprate (Sigma, MO) as a substrate, dissolved in dimethylsulfoxide (5 mMstock solution) and diluted in 4% Triton X-100, 1% SDS to a finalconcentration of 1 mM. For the assay, 20 mL of protein solution wasadded to a mixture of 25 mL of the 1 mM substrate solution and 80 mL of50 mM Tris pH 8.5. The enzyme activity was monitored at 405 nm in 6 secinterval for a period of 10 min. Esterase activity was expressed as DODmin⁻¹ mg⁻¹ protein.

Insect Bioassay

The protein was also assayed for activity against larvae of Diabroticavirigifera (Western corn rootworm) by overlaying the test sample on anagar diet similar to that described previously (Marrone et al., J. Econ.Entom. 78, 290-3 (1985)). Proteins to be tested were diluted in 25 mMTris/HCl pH 7.5 and overlayed on the diet surface. Neonate larvae wereallowed to feed on the diet and mortality and growth stunting wereevaluated after 6 days.

IgE Binding Epitopes on Patatin

A panel of eighty-nine overlapping peptides representing the amino acidsequence of patatin were synthesized to determine the regionsresponsible for IgE binding. Each peptide was 10 amino acids long andconsisted of 6 amino acid overlap between the consecutive peptides. Thepeptides were evaluated for IgE binding with five different potatoallergic patient sera. Patatin has 3 major epitopes. These major IgEbinding regions represent amino acids 184-193, 188-197, 269-278 and360-369. Other minor IgE binding regions represent amino acids 104-113,138-147 and 316-325. The amino acids essential for IgE binding in eachmajor and minor epitopes were determined by synthesizing peptides withsingle amino acid changes at each position by individually substitutingan alanine residue at each non-alanine position in the epitopes. Theresulting alanine substituted peptides were evaluated for IgE binding.Result effective substitutions were identified by a reduction in IgEbinding with respect to the non-substituted peptide sequence. It wasvery interesting to note that all the epitopes contained a Tyr residueand substitution of this Tyr for Ala or Phe eliminated IgE binding.

Enzyme and Bioactivity

The Tyr residues identified to be critical for IgE binding in each ofthe epitopes were substituted with Phe either individually or in concertusing site-directed mutagenesis. All the variants were expressed inPichia pastoris and assessed for enzyme activity and insecticidalactivity. The variants included Y106F, Y129F, Y185F, Y193F, Y270F,Y316F, Y362F, Y185F/Y193F, Y185F/Y193F/Y270F/Y316F/Y362F (5-Tyr) andY106F/Y129F/Y185F/Y193F/Y270F/Y316F/Y362F (7-Tyr). All the variantsmaintained enzyme activity. The 5-Tyr and 7-Tyr variants were thenassessed for insecticidal activity by overlaying protein (200 ppm finalconcentration). The proteins caused significant stunting of the larvalgrowth as measured by the weight of the larvae after 6 days with the5-Tyr variant showing higher insecticidal activity compared to the 7-Tyrand wild type proteins. The 7-Tyr variant was unstable upon long termstorage at 4° C. and thus was not pursued further.

Immunoblotting

In order to test if the glycan moiety on patatin was important forbinding of IgE, Pat17 was expressed in E. coli to produce anonglycosylated protein and in corn to produce a plant glycosylatedprotein. The 5-Tyr variant was also expressed in E. coli to assess theindividual contribution of the linear epitopes without the glycan moietyon the protein. The proteins were tested for binding to IgE using serafrom five patients with allergy to potatoes and sera from one patientwith allergies to many things but no allergy to potatoes. Proteins fromboth corn and E. coli were purified to homogeneity. These proteins weretransferred to PVDF membrane (Millipore, Mass.) and subsequently probedwith sera from patients with and without allergy to potatoes. AHis-tagged glyphosate oxidase control was included in all the studies toverify that the His-tag did not affect the binding of IgE. Serumobtained from patient HS-07 (no allergy to potatoes) did not bind Pat17expressed in E. coli but showed good binding to Pat17 from corn and alsoa protein at the same molecular weight in potato extract. It isinteresting to note that this sera also showed strong binding to anotherprotein (>46 kDa) in the potato. Sera from patients HS-01, HS-02, HS-03,HS-05 (allergy to potatoes) shows strong binding to Pat17 expressed incorn, but very weak to no binding to Pat17 produced in E. coli. Also,the sera from patients HS-01, HS-2, HS-03 and HS-05 bound to a proteinof similar molecular weight in the potato extract. Sera from patientsHS-01, HS-02 and HS-03 also showed binding to another protein in potatoextract of a lower molecular weight (<30 kDa). Serum obtained frompatient HS-06 (allergic to potatoes) showed very strong binding to wildtype patatin expressed in both corn and E. coli but weaker binding tothe 5-Tyr variant expressed in E. coli. Sera from HS-06 also showed verystrong binding to a protein in potato extract with similar molecularweight as patatin. The sera from all the patients showed no binding toHis-tagged glyphosate oxidase indicating that the His-tag does not bindIgE. These results strongly suggest that the glycan moiety on Pat17 isresponsible for IgE binding in some potato allergic patients and linearepitopes also contribute to the antigenicity of patatin.

Example 20

Alternative Nucleic Acid and Protein Sequences

For future variations of the patatin protein, sequences showing highsimilarity to the sequences disclosed herein could be used in producingdeallergenized patatin proteins and permuteins. For example, a BLASTsearch (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410, 1990) can beperformed to identify additional patatin sequences. Sources other thanthose disclosed herein can be used to obtain a patatin nucleic acidsequence, and the encoded patatin protein. Furthermore, subunitsequences from different organisms can be combined to create a novelpatatin sequence incorporating structural, regulatory, and enzymaticproperties from different sources.

Example 21

Nucleic Acid Mutation and Hybridization

Variations in the nucleic acid sequence encoding a patatin protein maylead to mutant patatin protein sequences that display equivalent orsuperior enzymatic characteristics when compared to the sequencesdisclosed herein. This invention accordingly encompasses nucleic acidsequences which are similar to the sequences disclosed herein, proteinsequences which are similar to the sequences disclosed herein, and thenucleic acid sequences that encode them. Mutations can includedeletions, insertions, truncations, substitutions, fusions, shuffling ofsubunit sequences, and the like.

Mutations to a nucleic acid sequence can be introduced in either aspecific or random manner, both of which are well known to those ofskill in the art of molecular biology. A myriad of site-directedmutagenesis techniques exist, typically using oligonucleotides tointroduce mutations at specific locations in a nucleic acid sequence.Examples include single strand rescue (Kunkel, T. Proc. Natl. Acad. Sci.U.S.A., 82: 488-492, 1985), unique site elimination (Deng and Nickloff,Anal. Biochem. 200: 81, 1992), nick protection (Vandeyar, et al. Gene65: 129-133, 1988), and PCR (Costa, et al. Methods Mol. Biol. 57: 31-44,1996). Random or non-specific mutations can be generated by chemicalagents (for a general review, see Singer and Kusmierek, Ann. Rev.Biochem. 52: 655-693, 1982) such as nitrosoguanidine (Cerda-Olmedo etal., J. Mol. Biol. 33: 705-719, 1968; Guerola, et al. Nature New Biol.230: 122-125, 1971) and 2-aminopurine (Rogan and Bessman, J. Bacteriol.103: 622-633, 1970), or by biological methods such as passage throughmutator strains (Greener et al. Mol. Biotechnol. 7: 189-195, 1997).

Nucleic acid hybridization is a technique well known to those of skillin the art of DNA manipulation. The hybridization properties of a givenpair of nucleic acids is an indication of their similarity or identity.Mutated nucleic acid sequences can be selected for their similarity tothe disclosed patatin nucleic acid sequences on the basis of theirhybridization to the disclosed sequences. Low stringency conditions canbe used to select sequences with multiple mutations. One may wish toemploy conditions such as about 0.15 M to about 0.9 M sodium chloride,at temperatures ranging from about 20° C. to about 55° C. Highstringency conditions can be used to select for nucleic acid sequenceswith higher degrees of identity to the disclosed sequences. Conditionsemployed may include about 0.02 M to about 0.15 M sodium chloride, about0.5% to about 5% casein, about 0.02% SDS and/or about 0.1%N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, attemperatures between about 50° C. and about 70° C. More preferably, highstringency conditions are 0.02 M sodium chloride, 0.5% casein, 0.02%SDS, 0.001 M sodium citrate, at a temperature of 50° C.

Example 22

Determination of Homologous and Degenerate Nucleic Acid Sequences

Modification and changes can be made in the sequence of the proteins ofthe present invention and the nucleic acid segments which encode themand still obtain a functional molecule that encodes a protein withdesirable properties. The following is a discussion based upon changingthe amino acid sequence of a protein to create an equivalent, orpossibly an improved, second-generation molecule. The amino acid changescan be achieved by changing the codons of the nucleic acid sequence,according to the codons given in Table 10.

TABLE 10 Codon degeneracies of amino acids One Amino acid letter Threeletter Codons Alanine A Ala GCA GCC GCG GCT Cysteine C Cys TGC TGTAspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAG Phenylalanine FPhe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine H His CAC CATIsoleucine I Ile ATA ATC ATT Lysine K Lys AAA AAG Leucine L Leu TTA TTGCTA CTC CTG CTT Methionine M Met ATG Asparagine N Asn AAC AAT Proline PPro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGACGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine T Thr ACA ACCACG ACT Valine V Val GTA GTC GTG GTT Tryptophan W Trp TGG Tyrosine Y TyrTAC TAT

Certain amino acids can be substituted for other amino acids in aprotein sequence without appreciable loss of enzymatic activity. It isthus contemplated that various changes can be made in the peptidesequences of the disclosed protein sequences, or their correspondingnucleic acid sequences without appreciable loss of the biologicalactivity.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle, J. Mol. Biol., 157: 105-132,1982). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics. These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2);glutamate/glutamine/aspartate/asparagine (−3.5); lysine (−3.9); andarginine (−4.5).

It is known in the art that certain amino acids can be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biologically functional protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those within ±1 are more preferred, and those within ±0.5 aremost preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 (Hopp, T. P., issued Nov. 19, 1985) states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. The following hydrophilicity values have beenassigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate(+3.0±1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine/histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine/isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4).

It is understood that an amino acid can be substituted by another aminoacid having a similar hydrophilicity score and still result in a proteinwith similar biological activity, i.e., still obtain a biologicallyfunctional protein. In making such changes, the substitution of aminoacids whose hydropathic indices are within ±2 is preferred, those within±1 are more preferred, and those within ±0.5 are most preferred.

As outlined above, amino acid substitutions are therefore based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine, andisoleucine. Changes which are not expected to be advantageous may alsobe used if these resulted in functional patatin proteins.

Example 23

Production of Patatin Proteins and Permuteins in Plants

Plant Vectors

In plants, transformation vectors capable of introducing nucleic acidsequences encoding patatin proteins and permuteins are easily designed,and generally contain one or more nucleic acid coding sequences ofinterest under the transcriptional control of 5′ and 3′ regulatorysequences. Such vectors generally comprise, operatively linked insequence in the 5′ to 3′ direction, a promoter sequence that directs thetranscription of a downstream heterologous structural nucleic acidsequence in a plant; optionally, a 5′ non-translated leader sequence; anucleic acid sequence that encodes a protein of interest; and a 3′non-translated region that encodes a polyadenylation signal whichfunctions in plant cells to cause the termination of transcription andthe addition of polyadenylate nucleotides to the 3′ end of the mRNAencoding the protein. Plant transformation vectors also generallycontain a selectable marker. Typical 5′-3′ regulatory sequences includea transcription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal. Vectors for plant transformation have beenreviewed in Rodriguez et al. (Vectors: A Survey of Molecular CloningVectors and Their Uses, Butterworths, Boston., 1988), Glick et al.(Methods in Plant Molecular Biology and Biotechnology, CRC Press, BocaRaton, Fla., 1993), and Croy (Plant Molecular Biology Labfax, Hames andRickwood (Eds.), BIOS Scientific Publishers Limited, Oxford, UK., 1993).

Plant Promoters

Plant promoter sequences can be constitutive or inducible,environmentally- or developmentally-regulated, or cell- ortissue-specific. Often-used constitutive promoters include the CaMV 35Spromoter (Odell, J. T. et al., Nature 313: 810-812, 1985), the enhancedCaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter (Richins etal., Nucleic Acids Res. 20: 8451-8466, 1987), the mannopine synthase(mas) promoter, the nopaline synthase (nos) promoter, and the octopinesynthase (ocs) promoter. Useful inducible promoters include promotersinduced by salicylic acid or polyacrylic acids (PR-1, Williams, S. W. etal, Biotechnology 10: 540-543, 1992), induced by application of safeners(substituted benzenesulfonamide herbicides, Hershey, H. P. and Stoner,T. D., Plant Mol. Biol. 17: 679-690, 1991), heat-shock promoters (Ou-Leeet al., Proc. Natl. Acad. Sci. USA. 83: 6815-6819, 1986; Ainley et al.,Plant Mol. Biol. 14: 949-967, 1990), a nitrate-inducible promoterderived from the spinach nitrite reductase gene (Back et al., Plant Mol.Biol. 17: 9-18, 1991), hormone-inducible promoters (Yamaguchi-Shinozaki,K. et al., Plant Mol. Biol. 15: 905-912, 1990; Kares et al., Plant Mol.Biol. 15: 225-236, 1990), and light-inducible promoters associated withthe small subunit of RuBP carboxylase and LHCP gene families (Kuhlemeieret al., Plant Cell 1: 471, 1989; Feinbaum, R. L. et al., Mol. Gen.Genet. 226: 449-456, 1991; Weisshaar, B. et al., EMBO J. 10: 1777-1786,1991; Lam, E. and Chua, N. H., J. Biol. Chem. 266: 17131-17135, 1990;Castresana, C. et al., EMBO J. 7: 1929-1936, 1988; Schulze-Lefert etal., EMBO J. 8: 651, 1989). Examples of useful tissue-specific,developmentally-regulated promoters include the β-conglycinin 7Spromoter (Doyle, J. J. et al., J. Biol. Chem. 261: 9228-9238, 1986;Slighton and Beachy, Planta 172: 356-363, 1987), and seed-specificpromoters (Knutzon, D. S. et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624-2628, 1992; Bustos, M. M. et al., EMBO J. 10: 1469-1479, 1991; Lamand Chua, Science 248: 471, 1991; Stayton et al., Aust. J Plant.Physiol. 18: 507, 1991). Plant functional promoters useful forpreferential expression in seed plastids include those from plantstorage protein genes and from genes involved in fatty acid biosynthesisin oilseeds. Examples of such promoters include the 5′ regulatoryregions from such genes as napin (Kridl et al., Seed Sci. Res. 1:209-219, 1991), phaseolin, zein, soybean trypsin inhibitor, ACP,stearoyl-ACP desaturase, and oleosin. Seed-specific gene regulation isdiscussed in EP 0 255 378. Promoter hybrids can also be constructed toenhance transcriptional activity (Comai, L. and Moran, P. M., U.S. Pat.No. 5,106,739, issued Apr. 21, 1992), or to combine desiredtranscriptional activity and tissue specificity.

Plant Transformation and Regeneration

A variety of different methods can be employed to introduce such vectorsinto plant protoplasts, cells, callus tissue, leaf discs, meristems,etcetera, to generate transgenic plants, includingAgrobacterium-mediated transformation, particle gun delivery,microinjection, electroporation, polyethylene glycol mediated protoplasttransformation, liposome-mediated transformation, etcetera (reviewed inPotrykus, I. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205-225,1991). In general, transgenic plants comprising cells containing andexpressing DNAs encoding patatin proteins and permuteins can be producedby transforming plant cells with a DNA construct as described above viaany of the foregoing methods; selecting plant cells that have beentransformed on a selective medium; regenerating plant cells that havebeen transformed to produce differentiated plants; and selecting atransformed plant which expresses the protein-encoding nucleotidesequence.

Specific methods for transforming a wide variety of dicots and obtainingtransgenic plants are well documented in the literature (Gasser andFraley, Science 244: 1293-1299, 1989; Fisk and Dandekar, ScientiaHorticulturae 55: 5-36, 1993; Christou, Agro Food Industry Hi Tech, p.17, 1994; and the references cited therein).

Successful transformation and plant regeneration have been reported inthe monocots as follows: asparagus (Asparagus officinalis; Bytebier etal., Proc. Natl. Acad. Sci. U.S.A. 84: 5345-5349, 1987); barley (Hordeumvulgarae; Wan and Lemaux, Plant Physiol. 104: 37-48, 1994); maize (Zeamays; Rhodes, C. A. et al., Science 240: 204-207, 1988; Gordon-Kamm etal., Plant Cell 2: 603-618, 1990; Fromm, M. E. et al., Bio/Technology 8:833-839, 1990; Koziel et al., Bio/Technology 11: 194-200, 1993); oats(Avena sativa; Somers et al., Bio/Technology 10: 1589-1594, 1992);orchardgrass (Dactylis glomerata; Horn et al., Plant Cell Rep. 7:469-472, 1988); rice (Oryza sativa, including indica and japonicavarieties; Toriyama et al., Bio/Technology 6: 10, 1988; Zhang et al.,Plant Cell Rep. 7: 379-384, 1988; Luo and Wu, Plant Mol. Biol. Rep. 6:165-174, 1988; Zhang and Wu, Theor. Appl. Genet. 76: 835-840, 1988;Christou et al., Bio/Technology 9: 957-962, 1991); rye (Secale cereale;De la Pena et al., Nature 325: 274-276, 1987); sorghum (Sorghum bicolor;Casas, A. M. et al., Proc. Natl. Acad. Sci. U.S.A. 90: 11212-11216,1993); sugar cane (Saccharum spp.; Bower and Birch, Plant J. 2: 409-416,1992); tall fescue (Festuca arundinacea; Wang, Z. Y. et al.,Bio/Technology 10: 691-696, 1992); turfgrass (Agrostis palustris; Zhonget al., Plant Cell Rep. 13: 1-6, 1993); wheat (Triticum aestivum; Vasilet al., Bio/Technology 10: 667-674, 1992; Weeks, T. et al., PlantPhysiol. 102: 1077-1084, 1993; Becker et al., Plant J. 5: 299-307,1994), and alfalfa (Masoud, S. A. et al., Transgen. Res. 5: 313, 1996);Brassica (canola/oilseed rape) (Fry, J. Plant Cell Rep. 6: 321-325,1987); and soybean (Hinchee, M. Bio/Technol. 6: 915-922, 1988).

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations can be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related can be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention.

1. An isolated nucleic acid sequence encoding the protein of SEQ ID NO:247.
 2. The isolated nucleic acid sequence of claim 1, comprising SEQ IDNO:
 246. 3. A recombinant vector operatively linked in the 5′ to 3′orientation, comprising: a) a promoter that directs transcription of astructural nucleic acid sequence; b) a nucleic acid sequence encodingthe protein of SEQ ID NO: 247; and c) a 3′ transcription terminator. 4.The recombinant vector of claim 3, wherein the vector expresses aprotein exhibiting corn rootworm insect inhibitory activity and acyllipid hydrolase activity.
 5. A recombinant host cell comprising anucleic acid sequence encoding the protein of SEQ ID NO:
 247. 6. Therecombinant host cell of claim 5, wherein the protein of SEQ ID NO: 247exhibits corn rootworm insect inhibitory activity and acyl lipidhydrolase activity.
 7. A recombinant plant comprising a nucleic acidsequence encoding the protein of SEQ ID NO:
 247. 8. The recombinantplant of claim 7, wherein the protein of SEQ ID NO: 247 exhibits cornrootworm insect inhibitory activity and acyl lipid hydrolase activity.